Therapeutic uses for nitric oxide inhibitors

ABSTRACT

The present invention is based on the discovery that nitric oxide (NO) is an important growth regulator in an intact developing organism. In particular, the present invention relates to a method of increasing in a mammal a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, wherein the hematopoietic tissue is contacted with multiple doses of at least one inhibitor of NO, such as one or more inhibitors of nitric oxide synthase (NOS), thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation. The present invention also relates to a method of increasing a population of cells in S phase in a tissue of a mammal, comprising contacting the tissue with multiple doses of at least one inhibitor (one or more) of NO, such as an inhibitor of NOS. The invention also pertains to a method of regenerating tissue in an adult mammal comprising contacting a selected tissue (e.g., blood, skin, bone and digestive epithelium), or precursor cells of the selected tissue, with multiple doses of at least one inhibitor (one or more) of NO, thereby inhibiting differentiation and inducing proliferation of cells of the tissue.

RELATED APPLICATION(S)

[0001] This application is a continuation of International ApplicationNo. PCT/US00/13685, which designated the United States and was filed onMay 18, 2000, published in English, which is a continuation-in-part ofU.S. application Ser. No. 09/315,929, filed May 20, 1999, which is acontinuation-in-part of U.S. application Ser. No. 08/969,475, filed Nov.13, 1997, which claims the benefit of U.S. Provisional application No.60/030,690, filed Nov. 13, 1996, and the benefit of U.S. Provisionalapplication No. 60/045,411, filed May 2, 1997. The entire teachings ofthe above application(s) are incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] Work described herein was supported by Grant No. 5RONS32764 fromthe National Institutes of Health. The United States Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Organ development requires a tightly controlled program of cellproliferation followed by growth arrest and differentiation and, often,programmed cell death. The balance between the number of cell divisionsand the extent of subsequent programmed cell death determines the finalsize of an organ (reviewed by Bryant and Simpson, Quart. Rev. of Biol.,59:387-415 (1984); Raft, Nature, 356:397-400 (1992)). Although much ofthe cellular machinery that determines the timing of onset and cessationof cell division per se is well understood (reviewed by Hunter andPines, Cell, 79:573-582 (1994); Morgan, Nature, 374:131-134 (1995);Weinberg, Cell, 81:323-330 (1995)), little is known about the signalsthat cause discrete groups of cells and organs to terminate growth atthe appropriate cell number and size. A better understanding of thesignals involved provides possible targets for manipulating the cellularmachinery resulting in therapeutic benefits for a number of conditions.

SUMMARY OF THE INVENTION

[0004] The present invention is based on the discovery that nitric oxide(NO) is an important growth regulator in an intact developing organism.In particular, the present invention relates to a method of increasingin a mammal a population of hematopoietic cells (e.g., hematopoieticstem cells), including precursors to myeloid, lymphoid and erythroidcells, which are capable of undergoing normal hematopoiesis,differentiation and maturation in hematopoietic tissue, wherein thehematopoietic tissue is contacted with multiple doses of at least oneinhibitor of NO, such as multiple doses of one or more inhibitors ofnitric oxide synthase (NOS), thereby producing hematopoietic tissuehaving an increased population of hematopoietic stem cells which arecapable of undergoing normal hematopoiesis, differentiation andmaturation. In one embodiment, the present invention relates to a methodof increasing in a mammal a population of hematopoietic stem cells whichare capable of undergoing normal hematopoiesis, differentiation andmaturation in hematopoietic tissue, comprising contacting thehematopoietic tissue with two inhibitors of nitric oxide synthase,thereby producing hematopoietic tissue having an increased population ofhematopoietic stem cells which are capable of undergoing normalhematopoiesis, differentiation and maturation. The method can be carriedout in vivo or ex vivo. In addition, the method can be used to preventdifferentiation of erythroid cells and/or myeloid cells in the mammal.The method can further comprise contacting the hematopoietic tissue withat least one agent (e.g., a hematopoietic growth factor) which inducesdifferentiation of a selected hematopoietic stem cell population.

[0005] The present invention also relates to a method for treating amammal to increase a population of hematopoietic stem cells which arecapable of undergoing normal hematopoiesis, differentiation andmaturation in hematopoietic tissue of the mammal. In the method, thehematopoietic tissue of the mammal is contacted with multiple doses ofat least one inhibitor of NOS, thereby producing hematopoietic tissuehaving an increased population of hematopoietic stem cells which arecapable of undergoing normal hematopoiesis, differentiation andmaturation. In one embodiment, the present invention relates to a methodfor treating a mammal to increase a population of hematopoietic stemcells which are capable of undergoing normal hematopoiesis,differentiation and maturation in hematopoietic tissue of the mammal,comprising contacting the hematopoietic tissue of the mammal with twoinhibitors of nitric oxide synthase, thereby producing hematopoietictissue having an increased population of hematopoietic stem cells whichare capable of undergoing normal hematopoiesis, differentiation andmaturation. The method can further comprise contacting the hematopoietictissue with at least one agent which induces differentiation of aselected hematopoietic stem cell population.

[0006] In one embodiment of the method for treating a mammal to increasea population of hematopoietic stem cells which are capable of undergoingnormal hematopoiesis, differentiation and maturation in hematopoietictissue of the mammal, hematopoietic tissue which is to be transplantedis obtained, wherein the hematopoietic tissue to be transplanted can beobtained from the mammal being treated (autologous transplantation) orfrom another mammal (heterologous transplantation). The hematopoietictissue to be transplanted is contacted with multiple doses of at leastone inhibitor of NOS. The hematopoietic tissue which is to betransplanted is transplanted into the mammal being treated, therebyproviding the mammal with hematopoietic tissue having an increasedpopulation of hematopoietic stem cells which are capable of undergoingnormal hematopoiesis, differentiation and maturation. In one embodiment,two NOS inhibitors are used. The method can further comprise treatingthe mammal with an inhibitor(s) of NOS before or after transplanting thehematopoietic tissue. Alternatively, the method can further comprisetreating the mammal with an enhancer (one or more) of NOS before orafter transplanting the hematopoietic tissue.

[0007] The present invention also relates to a method of increasing apopulation of progenitor blood cells (e.g., red blood cells, white bloodcells) which are capable of undergoing normal hematopoiesis,differentiation and maturation comprising contacting progenitor cells(stem cells) of blood with multiple doses of at least one inhibitor ofNO (e.g., an inhibitor of NOS). In one embodiment, the progenitor bloodis contacted with two inhibitors of NOS.

[0008] The present invention also relates to a method of increasing apopulation of dividing cells in a tissue of a mammal comprisingcontacting the cells with multiple doses of at least one inhibitor ofnitric oxide. In one embodiment, the present invention also relates to amethod of increasing a population of cells in S phase in a tissue of amammal, comprising contacting the tissue with multiple doses of at leastone inhibitor of NO, such as an inhibitor of NOS. In one embodiment, themethod results in an increase in the size of an organ in which thetissue is occurs. Furthermore, as described herein the cells in S phasecan be used in gene therapy.

[0009] The present invention also relates to a method of decreasing apopulation of cells in S phase in a tissue of a mammal and inducingdifferentiation of the cells, comprising contacting the tissue with anenhancer(s) of NO, such as an enhancer of NOS. In one embodiment, themethod results in a decrease in the size of an organ with which thetissue is associated.

[0010] The present invention also relates to a method of coordinatingdevelopmental decisions of a cell type in a mammal, comprisingintroducing NO into the cell type or a precursor of the cell type,thereby inhibiting proliferation of the cell type or a precursor of thecell type and inducing differentiation of the cell type or a precursorof the cell type.

[0011] A method of inducing differentiation in a mammalian cellpopulation comprising contacting the cell population with NO or a NOenhancer is also encompassed by the present invention.

[0012] The invention also pertains to a method of regenerating tissue inan adult mammal comprising contacting a selected tissue (e.g., blood,skin, bone and digestive epithelium), or precursor cells of the selectedtissue, with multiple doses of at least one inhibitor of NO, therebyinhibiting differentiation and inducing proliferation of cells of thetissue, then contacting the selected tissue with a compound (e.g.,nitric oxide, a growth factor or a combination of both) which inhibitsproliferation and induced differentiation. In one embodiment, the methodinvolves repopulating an organ or tissue (e.g., muscle or nerve fiber)comprised of normally nondividing cells by contacting a selected organor tissue, or precursor cells of the selected organ or tissue, withmultiple doses of at least one inhibitor of NO, thereby inhibitingdifferentiation and inducing proliferation of cells of the organ ortissue, then contacting the selected organ or tissue with a compoundwhich inhibits proliferation and induced differentiation.

[0013] The invention also encompasses a method of producing asubpopulation of hematopoietic cells. In the method, hematopoietictissue is contacted with multiple doses of at least one inhibitor ofNOS, thereby producing hematopoietic tissue having an increasedpopulation of hematopoietic stem cells which are capable of undergoingnormal hematopoiesis, differentiation and maturation; and at least oneagent (e.g., a hematopoietic growth factor) selected to induce specificdifferentiation of the hematopoietic stem cell population, therebyproducing a subpopulation of hematopoietic cells. In a particularembodiment, the hematopoietic tissue is contacted with two inhibitors ofNOS.

[0014] Identification of NO as an important growth regulator in anorganism provides for various therapeutic applications in humans andother mammals.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Results of the work described herein have shown that atranscellular messenger (nitric oxide (NO)) plays a critical role intissue differentiation and organism development. NO regulates thebalance between cell proliferation and cell differentiation in theintact developing organism. Increased production of NO permits cessationof cell division and subsequent differentiation of cell in a tissue,whereas removal of the NO-mediated growth arrest promotes cell division.

[0016] Accordingly, the present invention relates to a method ofincreasing in a mammal a population of hematopoietic cells (e.g.,hematopoietic stem cells), including precursors to myeloid, lymphoid anderythroid cells, which are capable of undergoing normal hematopoiesis,differentiation and maturation in hematopoietic tissue, by contactingthe hematopoietic tissue with multiple doses of at least one inhibitor(one or more) of NO, such as an inhibitor of NOS. As defined herein“hematopoietic tissue” is tissue involved in hematopoiesis. e.g., bonemarrow, peripheral blood, umbilical cord vein blood, fetal liver, andlong term hematopoietic cell culture.

[0017] The present invention includes a method for treating a mammal toincrease a population of hematopoietic stem cells which are capable ofundergoing normal hematopoiesis, differentiation and maturation inhematopoietic tissue of the mammal, in which the hematopoietic tissue ofthe mammal is contacted with multiple doses of at least one inhibitor ofNOS. The invention also pertains to a method of producing asubpopulation of hematopoietic cells by contacting hematopoietic tissuewith multiple doses of at least one inhibitor of NOS, thereby producinghematopoietic tissue having an increased population of hematopoieticstem cells which are capable of undergoing normal hematopoiesis,differentiation and maturation; and at least one agent selected toinduce specific differentiation of the hematopoietic stem cellpopulation, thereby producing a subpopulation of hematopoietic cells. Ina particular embodiment, two inhibitors of NO, such as two inhibitors ofNOS, are used in the methods. For example, a combination of L-NAME andETU can be contacted with the hematopoietic tissue to increase apopulation of hematopoietic stem cells in a mammal, to treat a mammal toincrease a population of hematopoietic stem cells in hematopoietictissue in a mammal or to produce a subpopulation of hematopoietic cells.

[0018] The present invention also relates to a method of increasing apopulation of progenitor blood cells comprising contacting progenitorcells of blood with multiple doses of at least one inhibitor (one ormore) of NO (e.g., inhibitor of NOS). In one embodiment, the presentinvention relates to a method of increasing a population of progenitorblood cells comprising contacting progenitor cells of blood with twoinhibitors of NOS. The sources of progenitor cells of blood include, forexample, bone marrow, peripheral blood, umbilical cord vein blood, fetalliver, and long term hematopoietic cell culture. Using the method of thepresent invention red blood cells and white blood cells (e.g.,granulocytes (neutrophils, basophils, eosinophils), monocytes,lymphocytes) can be increased.

[0019] The present invention also relates to a method of increasing apopulation of dividing cells in a tissue of a mammal comprisingcontacting the cells with multiple doses of at least one inhibitor ofNO. In one embodiment, the present invention can also be used toincrease a population of cells (targeted cells) in S phase in a tissueof a mammal relative to a similar tissue in an untreated mammal, bycontacting the tissue with multiple doses of at least one inhibitor ofNO, such as an inhibitor of NOS. In one embodiment, the method resultsin an increase in the size of an organ with which the tissue isassociated. Conversely, the present invention can also be used todecrease a population of cells in S phase in a tissue of a mammal andinducing differentiation of the cells, comprising contacting the tissuewith at least one enhancer of NO, such as an enhancer of NOS. In oneembodiment, the method results in a decrease in the size of an organwith which the tissue is associated. Furthermore, as described hereinthe cells in S phase can be used in gene therapy.

[0020] The present invention also relates to a method of coordinatingdevelopmental decisions of a cell type in a mammal, comprisingintroducing NO into the cell type or a precursor of the cell type,thereby inhibiting proliferation of the cell type or a precursor of thecell type and inducing differentiation of the cell type or a precursorof the cell type. A method of inducing differentiation in a mammaliancell population comprising contacting the cell population with NO or aNO enhancer is also encompassed by the present invention.

[0021] The invention also pertains to a method of regenerating tissue inan adult mammal. The method comprises contacting a selected tissue withmultiple doses of at least one inhibitor of NO, thereby inhibitingdifferentiation and inducing proliferation of cells of the tissue, thencontacting the selected tissue with a compound which inhibitsproliferation and induces differentiation of the proliferated cells tocells characteristic of the tissue. In one embodiment, the methodinvolves repopulating an organ or tissue (e.g., muscle or nerve fiber)having normally nondividing cells comprising contacting a selected organor tissue with multiple doses of at least one inhibitor(s) of NO,thereby inhibiting differentiation and inducing proliferation of cellsof the organ or tissue, then contacting the selected organ or tissuewith a compound which inhibits proliferation and induces differentiationof the proliferated cells to cells characteristic of the organ ortissue. Compounds which inhibit proliferation and induce differentiationinclude NO, an enhancer of NO and a growth factor. One or more thesecompounds can be used to inhibit proliferation and inducedifferentiation.

[0022] Tissue which can be regenerated using the methods describedherein include blood, skin, bone and digestive epithelium, nerve fiber,muscle, cartilage, fat or adipose tissue, bone marrow stroma andtendons.

[0023] The methods described herein can further comprise the step ofcontacting the hematopoietic tissue target cells (e.g., bone marrow)with at least one agent which induces differentiation of a selectedhematopoietic stem cell population to a particular cell type (e.g.,erythrocytes, macrophages, lymphocytes, neutrophils and platelets). Forexample, in the embodiment wherein a mammal is treated to increase apopulation of hematopoietic stem cells in the hematopoietic tissue ofthe mammal by contacting the hematopoietic tissue of the mammal withmultiple doses of at least one inhibitor of NOS, the increasedpopulation of hematopoietic tissue can be contacted with an agent, suchas a hematopoietic growth factor, which will cause or promotedifferentiation of the cells of a particular cell type. Agents (e.g.,such as hemopoietic growth factors) which can be used in the methods ofthe present invention to induce differentiation of the increased orexpanded number of cells produced by contacting cells with a NOSinhibitor include, for example, erythropoietin, G-CSF, GM-CSF andinterleukins such as IL-1, IL-2, IL-3 and IL-6. Alternatively, themethods described herein can further comprise the step of contacting thehematopoietic tissue with at least one agent which further induces ormaintains proliferation of the selected hematopoietic stem cellpopulation to a particular cell type (e.g., erythrocytes, macrophages,lymphocytes, neutrophils and platelets). Inhibitors of NO for use in thepresent invention include, for example, NO scavengers such as2-phenyl-4,4,5,5-tetraethylimidazoline-1-oxyl-3-oxide (PTIO),2-(4-carboxyphenyl)-4,4,5,5-tetraethylimidazoline-1-oxyl-3-oxide(Carboxy-PTIO) and N-methyl-D-glucamine dithiocarbamate (MGD); and NOSinhibitors such as N-nitro-L-arginine methyl-ester (L-NAME),N-monomethyl-L-arginine (L-NMMA), 2-ethyl-2-thiopseudourea (ETU,),2-methylisothiourea (SMT), 7-nitroindazole, aminoguanidine hemisulfateand diphenyleneiodonium (DPI).

[0024] In the methods of the present invention, multiple doses of atleast one inhibitor of NO (e.g., NOS inhibitors) can be used. When morethan one inhibitor is used in the methods of the present invention, theinhibitors can be the same or different. In a particular embodiment, twoinhibitors of NO, such as two inhibitors of NOS (e.g., L-NAME and ETU),are used in the methods of the present invention.

[0025] Furthermore, in the methods of the present invention, the NOinhibitor(s) can be administered in a single dose or in multiple doses.As used herein, “multiple doses” refers to at least two doses of atleast one inhibitor of NO. The multiple doses can be administered in aday or over a period of days (e.g., a period of about 2 days to a periodof about 15 days or months). For example, the NO inhibitor(s) can beadministered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15days. Inone embodiment, a mixture of two NO inhibitors (e.g., L-NAME andETU) are administered to the mammal or contacted with the cells twice aday for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In aparticular embodiment, L-NAME and ETU are administered to the mammal orcontacted with the cells twice a day for 9 days.

[0026] In the methods of the present invention, one or more enhancers ofNO can be used. Enhancers of NO include, for example, NOS enhancers, andNO donors such as sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (SNOG,GSNO), diethylamine NONOate (DEA/NO), DETA/NO (NOC-18),3-morpholinosydnonimine (SIN-i) and spermine NONOate (Sper/NO).

[0027] NO is a diffusible multifunctional second messenger that has beenimplicated in numerous physiological functions in mammals, ranging fromdilation of blood vessels to immune response and potentiation ofsynaptic transmission (Bredt and Snyder, Annu. Rev. Biochem., 63:175-195(1994); Nathan and Xie, Cell, 78:915-918 (1994); Garthwaite and Boulton,Annu. Rev. Physiol., 57:683-706(1995)). NO is produced from arginine byNOS in almost all cell types. A group of three chromosomal genes, givingrise to numerous isoforms of NOS, have been cloned from mammalian cells(Knowles and Moncada, Biochem. J., 298:249-259 (1994); Wang and Marsden,Adv. Pharmacol., 34:71-90 (1995)), and recently a Drosophila NOS gene,whose coding structure resembles the gene for the mammalian neuronalisoform, has been isolated (Regulski and Tully, Proc. Natl. Acad. Sci.USA, 92:9072-9076 (1995)).

[0028] Cell division and subsequent programmed cell death in imaginaldiscs of Drosophila larvae determine the final size of organs andstructures of the adult fly. Results described herein show that NO isinvolved in controlling the size of body structures during Drosophiladevelopment. These results demonstrate that NOS is expressed at highlevels in developing imaginal discs. Inhibition of NOS in larvae causeshypertrophy of organs and their segments in adult flies, whereas ectopicexpression of NOS in larvae has the opposite effect. Blocking apoptosisin eye imaginal discs unmasks surplus cell proliferation and results inan increase in the number of ommatidia and component cells of individualommatidia. These results demonstrate the activity of NO as anantiproliferative agent during Drosophila development, controlling thebalance between cell proliferation and cell differentiation. Moreover,results shown here demonstrate that NO acts as a crucial regulator ofhematopoiesis after bone marrow (BM) transplantation. NO regulates thematuration of both the erythroid and myeloid lineages. These datademonstrate that manipulations of NOS activity and NO levels duringhematopoiesis can be used to alter (enhance or reduce) blood cellproduction. This is useful for preventive and therapeutic intervention.

[0029] During Drosophila development, the structure, size, and shape ofmost of the organs of the adult fly are determined in the imaginalstructures of the larvae (Cohen, Imaginal disc development, in TheDevelopment of Drosophila melanogaster, M. Bate and A. Martinez-Afias,eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),747-841 (1993); Fristrom and Fristrom, The metamorphic development ofthe adult epidermis, in The Development of Drosophila melanogaster, M.Bate and A. Martinez-Afias, eds. (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), 843-897 (1993)). Imaginal discs, specializedgroups of undifferentiated epithelial cells that are recruited duringembryogenesis, are formed in the first larval instar as integuments ofthe larval epidermis. Disc cells divide rapidly throughout the larvaldevelopment and cease proliferating at the end of the third instarperiod.

[0030] In leg, wing, and haltere discs, progression through the cellcycle stops in G2 phase 3-4 hours before puparium formation. It resumes15-18 hours later (12-14 hours after pupariation) and then stops againin a defined spatial pattern after 12-14 hours (10-14 hours of pupaldevelopment) (Fain and Stevens, Dev. Biol., 92:247-258 (1982); Gravesand Schubiger, Dev. Biol., 93:104-110 (1982); Schubiger and Palka, Dev.Biol., 123:145-153 (1987)). Although most of the dividing cells in thelate larvae and in the early pupae are already committed to their adultfate, they do not develop a fully differentiated phenotype until growtharrest is firmly established. Thus, cell proliferation is temporallyseparated from cell differentiation, which takes place later duringmetamorphosis. Experiments with transplanted imaginal discs suggest thatcessation of cell proliferation in these structures is controlled bymechanisms that, while intrinsic to the disc, are not completelycell-autonomous (Bryant and Schmidt, J. Cell Sci., Suppl. 13:169-189(1990); Cohen, Imaginal disc development, in The Development ofDrosophila melanogaster, M. Bate and A. Martinez-Afias, eds. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), 747-841(1993)). The signaling pathways that control coordinated temporarygrowth arrest in larvae and pupae and subsequent terminal growth arrestin pupae and adults are not known, but they probably involveintercellular and intracellular second messenger molecules which havenot yet been identified.

[0031] Transformation of imaginal precursors in adult structures duringfly metamorphosis involves transition from cell proliferation to celldifferentiation. Cessation of cell division is a necessary, although notsufficient, condition for cell differentiation to proceed. A temporarycytostasis occurs at the end of the larval period, and permanent arrestof cell division occurs during pupal development. NO, a diffusiblemessenger molecule, is capable of efficiently blocking cell division.Induction of NOS initiates a switch to growth arrest prior todifferentiation of cultured neuronal cells (Peunova and Enikolopov,Nature, 375:68-73 (1995)). Thus, NOS can act as a permissive factor,making the further development of the fully differentiated phenotypepossible. Results described herein show that NOS acts as anantiproliferative agent during normal Drosophila development, indicatingthat NO is an important growth regulator in the intact developingorganism.

[0032] Throughout larval development, there is a gradual andspatially-specific accumulation of NADPH-diaphorase activity indeveloping imaginal discs, reflecting an increase in overall NOScontent. At the time temporary cytostasis is being established inimaginal discs, NADPH-diaphorase staining becomes particularly intense,and it gradually decreases during prepupal and pupal development.Besides the imaginal discs, other structures with intenseNADPH-diaphorase staining include imaginal rings, histoblasts and thebrain. These structures undergo radical changes during metamorphosisbefore giving rise to adult organs. Their development includes periodsof rapid cell division alternating with periods of cytostasis, and thusmust employ mechanisms for coordinated cessation of DNA synthesis andcell division in a spatially defined pattern. Since NO can prevent celldivision and can diffuse and act within a limited volume, the ability ofNO to act to induce coordinated growth arrest during Drosophiladevelopment was considered. Indeed, if NO actively exerts itsantiproliferative activity during the development of imaginal discs,then inhibition of NOS before the temporary cytostasis is established atthe end of the larval period could lead to the reversal of the arrest ofcell division and induce additional divisions, which in turn could leadto increased size of structures of the body of the adult fly.Conversely, excessive or ectopic-production of NO in larvae could causepremature cessation of cell division and lead to a reduction in the sizeof the structures in the adults.

[0033] Both predictions were confirmed in experiments described herein,in which NOS activity was manipulated in the developing fly. NOSinhibition in larvae caused an increase in the number of cells in someparts of the adult body and an increase in their size, whereas ectopicexpression of the NOS transgene during development caused a decrease inthe number of cells in some structures in the adult and a decrease intheir size, probably by partial fusion and reduction. In the developingleg, the segments that were most often affected when NOS activity wasinhibited and the segments that were most often affected when theactivity was inhibited and the segments that were most often affectedwhen the acrivity was ectopically induced were nonoverlapping andcomplementary. Most importantly, their distribution matched thedistribution on NOS in the imaginal discs, thereby supporting thehypothesis that NO plays a causative role in growth arrest in normaldevelopment.

[0034] The antiproliverative properties of NO suggest that NOS acts indevelopment through its influence on DNA synthesis and cell division.The results described herein with BrdU incorporation in leg discs withelevated and diminished production on NO corroborate this position andsuggest a direct link between synthesis on NO, number of S-phase cells,and the final size of the organ. In accordance with this idea, in manyinstances no BrdU incorporation was observed in regions highly enrichedin NOS. The Mechanisms for the final size of the organ. In accordancewith this idea, in many instances no BrdU incorporation was observed inregions hightly enriched in NOS. The mechanisms for the NO-mediatedarrest of the cell cycle (both temporary and terminal) are not clear,but they likely involved the conventional cellular machinery for growtharrest, e.g., cell cycly-dependent kinases and their inhibitors.consistent with this, changes in expression of these proteins wereobserved when cultured cells were treated with NO. An intriguing featureof imaginal disc cells is that they stop dividing and accumulate in G2phase in the late third instar, preceding the perid of temporarycytostasis (Fain and Stevens, Dev. Biol., 92:247-358 (1982); Graves andSchubiger, Dev. Biol., 93:104110 (1982); Schubiger and Palka, Dev.Biol., 123:145-153 (1987). This parallels a tendency of NO-treated(Peunova and Enikolopov, Nature, 375:68-73 (1995)) and NGF-treated(Buchkovich and Ziff, Mol. Bio. Cell, 5:225-241 (1994)) PC12 cells toaccumulate in G2 phase. Interestingly, imaginal discx are released fromthe G2 block and reenter S phase 12-15 hours after population, at thetime when diaphorase stainging is diminished to low levels in adultflies. These correlations between imaginal discs cells and No-treatedcells support the idea that NO can be a major inducer of cytostasis inthe cells of imaginal discs in the prepupal stage.

[0035] The final number of cells in an organ or a segment is determinedboth by cell multiplication and cell death, which the forming structuresof the fly undergo as a normal step in development (especially at thelate stages of pupal development). Results described herein indicatethat the changes in the size of the leg segments after manipulation ofNOS activity correlated directly with the changes in DNA synthesis andthe number of dividing cells. Furthermore, no significant changes inapoptosis were detected in the larval and prpupal leg discs afterinhibitions or ectopic expression of NOS, compared with the controldiscs, when cell deth was monitored by acridine-orange staining or bythe TUNEL assay. This suggests that it is cell multiplation, rather thanchanges in programmed cell death that leads to the changes in the sizeof the appendage.

[0036] On the other hand, apoptotic deth may conceal excessive cellproliferation in other developing organs. Teh effect of the absence ofproogrammed cell death on potential excessive cell proliferation wasalso assessed. Transgenic flies were used in which programmed cell deathin the developing eye was suppressed by recombinant p35, an inhibitor ofapptosis, to reveal excessive proliferation after NOS inhibition. Underthese circumstances, several celltypes and structures areoverrepresented, the most noticeable change being an overall increase ofthe size of the eye due to the increased number of ommatidia. Inaddition, other cell types (e.g., secondary and tertiary pigment cells,cone cells, and cells of the bristles) proliferated after NOS inhibitionto levels higher that those achieved by blocking apoptosis by p35 (Hayet al., Dev., 120:2121-2129 (1994)). These data demonstrate that theremoval of suppressive influence of NO leads to an increase in the sizeof te adult organ, unless this effect is masked by programmed celldeath, and indicate that final cell number in the adult organ is underdual control by both cell proliferation and programmed cell death.Furthermore, these data provide independent support for thehypothesisthat NO directly regulates cell number during developmet.

[0037] After inhibition of NOS with either of two stucurally unrelatedcompounds, excessive groth was observed in most of the structures of theadult flies that derive from imaginal discs and histoblasts, to varyingextents for different organs. The most obvious changes were observed inthe segments of the legs whose primordia showed the highest levels ofNOS. There did not appear to be any substantial number of instances inwhich a dupication of a larger structure (for example, segments of thelegs or wings) occurred. This indicates that extra proliferation ofcells under the influence of NOS inhibitors occurs after thedevelopmental fate is determined for most of the cells in the imaginaldiscs. This suggests that in most cases NO may be more important for theinduction of growth arrest and subsequent differentiation of alreadycommited cells than for the developmental commitment and establishmentof the cell identity in the embryo or larvae.

[0038] Only some of the axes of the developing structures were affectedby manipulations of the NOS activity. For instance, in developing legsonly the anteroposterior and the dorsoventral axes, but not theproximodistal axis, were affected by inhibition of NO production, Incontrast, when NOS was ectipically expressed, only the proximodistalaxis was affected. These results suggest that a gradient of NO may beinvolved in the process of establishing the polarity of the axes of thedeveloping organ.

[0039] Thus these results demonstrate that inhibition of NOS in larvaeleads to enlargement of organs in adults and, conversely, that ectopicexpression of NOS in larvae leads to a reduction in the size of organsin adults. Also, the distribution of affected segments in the adult legcorresponds to the distribution of NOS in the larvae, and the changes insegment size can be directly correlated to changes in DNA synthesis inimaginal discs after manipulations of NOS activity. The increased cellproliferation that occurs in response to NOS inhibition is masked insome structures by programmed cell death, and it can be revealed bysuppressing apoptosis. Taken together, these results demonstrate thatactivation of NOS is a crucial step in Drosophila development. Theyconfirm that NO acts as an antiproliferative agent during celldifferentiation and organism development and controls the cell number inan intact developing organism.

[0040] NOS expression can be induced to high levels in a large number oftissues and cell types by appropriate stimulation (Bredt and Snyder,Annu. Rev. Biochem., 63:175-195 (1994); Forstermann et al., Adv.Pharmacol., 34:171186 (1995)). In most cases, the pattern of NOSdistribution in a developing organism differs strongly form thedistribution in the adult organism. Furthermore, transient elevation ofNOS expression in a given tissue often coincides with the cessation ofdivision of committed precursor cells. The developing mammalian brainprovides an especially apt demonstration of this (Bredt and Snyder,Neuron, 13:301-313 (1994); Blottner et al., Histochem. J., 27:785-811(1995)). A strong elevation of NOS activity in the developing cerebralcortical plate and hippocampus at days 15-19 of prenatal developmentcorrelates with the timecourse of cessation of precursor cellsproliferation, tight growth arrest, and cell differentiation; notably,NOS activity goes down after the proiferation of committed neuronalprcursors is completed. NOS levels are also transiently increased indeveloping lungs, bones, blood vessels, and nervous system (Blottner etal., Histochem. J., 27:785-811 (1995); Collin-osdoby et al., J. CellBiochem., 56;399-408 (1995); Cramer et al., J. Comp. Neurol.,353:306-316 (1995); Shaul, Adv. Pediatr., 42:367-414 (1995); Wetts etal., Dev. Dyn., 202:215-228 (1995). Elsewhere, NOS activity is greatlyelevated in regernarating tissue when cessation of cell division iscrucial for prevention of the unreggulated growth (Roscams etal.,Neuron, 13:289-2y9 (1994); Blottner et al., Histochem. J.,27:785-811 (1995); Decker and Obolenskaya, J. Gastroenterol. Hepatol, 10Supp 1:2-7 (1995); Horetlano et al., Hepat. 21:776-786 (1995)). In allthese cases, a transient elevation of NOS activity might trigger aswitch from proliferation to growth arrest and differentiation, thuscontributing to the proper morphogenesis of the tissue and the organ.

[0041] Results described herein support the position that production ofNO is required during embroyonic development and during tissueregeneration in the adult organism for the proper control of cellproliferation. The antiproliferative properties of NO are particularlyimportant in situations in which terminal differentiation of committedcells is temporallly departed from cell proliferation and is strictlydependent on cessation of cell division. Given the multiplicity of theNOS isoforms and their overlapping tissue distribution, it isconceivable that any group of cells in the embryo and fetus can beexposed to NO action. Furthermore, recent data showing that NO can betransferred within the organism by hemoglobin (Jia et al., Nature,380:221226 (1996) raise the possibility that a developing mammalianembryo can be also supplied with No exogenously by the mother.

[0042] NO is a readily diffusible molecule, and it may there exert itsantiproliferative properties not only in the cell that produces it butin the neighboring cells as well (gally et al., Proc. Natl. Acad. Sci.USA, 87:3547-3551 (1990). This property is important when one considersmechanisms for the coordinated development of a group of neighboringcells committed to form a particular structure. These cells have togenerate an intrinsic signal that tells them to stop dividing in acoordinated fashion after they have reached a certain number. Thiscooperation and coordiantion is achieved in many instances by tightlycontrolled paracrine regulation, which involves signaling betweenadjacent cells via gap junctions or secreted proteins. Results describedherein show that yet another way of coordinating developmental decisionsin groups of cells is by diffusible antiproliferative second messengermolecules, which can spread without a need for surface receptors orspecialized systems for secretion and exert their influence within alimited domain. An efficient source of readily diffusible molecules mayinduce sychronized changes in the adjacent cells within a limited volumeof a tissue. Moreover, several adjacent cells producing easilydiffusible antiproliferative messenger molecules may share the totalpool of these molecules produced by the neighbors as well as bythemselves. If a particular threshold level of a signal is needed toinitiate a signaling chain that eventually leads to growth arrest, thenthe cells in this group could stop dividing when a certain number ofcells and, therefore, a certain local concentration of messengermolecules, is reached. In this way, by organizing groups of cells infunctional clusters and coordinating their decisions on proliferationand differentiation, No instruct the developing structures to terminatetheir growth when they attain the appropriate size and shape, and, thus,participate in tissue and organ morphogenesis.

[0043] As also described herein, the role of NO in hematopoiesis wasexamined. To demonstrate the presence of NOS in the bone marrow (BW)cells, BM from adult mice was tested for the NDPH-disphorase activity ofNOS (which reflects the distribution of the total enzyme activity in atissue). It was found that BM contains a substantial proportion of cells(up to 12%) with strong diaphorasc staining. The morphology of theNADPH-diaphorase cells suggests that they are largely of thegranulocyte-macrophage lineage at different stages of differentiation.This is in accordance with numerous data showing that NOS is present inthe cells of the myeloid lineage, and can be induced to high levels byappropriate stimulation.

[0044] A mouse model of syngeneic BM transfer was used to evaluate therole of NO in hematopoiesis. Mice were irradiated to inhibithematopoiesis in the recipient animal, BM was transplanted fromsyngeneic animals, and the animals were treated with specific NOSinhibitors. This procedure permits the proliferation, differentiationand survival of only the transplanted cells. To study the changes inhematopoiesis introduced by NOS inhibitors, the colonies in the spleenwere monitored to test the differentiation of erythroid cells, and theformation of colonies on the membranes placed in the peritoneal cavityof the recipients were monitored to test the differentiation of cells ofthe granulocyte-macrophage lineage. The role of NO on hematopoiesis wastested by injecting the animals with the specific and structurallyunrelated NOS inhibitors L-nitroarginine methy ester (L-NAME), and2-ethyl-2-thiopseudourea (ETU). The inactive enanitomer D-NAME was usedas a control. Animals were sacrificed and the number and composition ofcolonies in the spleen (reflecting the cells which have undergoneerythroid differentiation) and colonies on the membranes (reflecting thecells that have undergone myeloid differentiation) were studied.

[0045] Taken together, the results of these studies indicate that NOmodulates hematopoiesis after BM transplantation. This confirms the roleof NO as a major regulatory factor in the organism controlling thebalance between proliferation and differentiation. This also shows thatmanipulation of NO levels may be used for therapeutic intervention toincrease the number of undifferentiated hematopoietic cells after BMtransplantation; change the ratio of cells undergoing erythroid ormyeloid differentiation; and interfere or suppress graft-versus-hostdisease, which is a major cause of mortality in patients undergoing BMtransplantation.

[0046] Most of the tissues and organs in the adult organism areconstantly undergoing regeneration and renovation, going through phasesof rapid proliferation, determination, growth arrest, differentiation,and often, programmed cell death. Many human diseases are caused byimproper or incomplete differentiation steps, resulting in the loss offunction of a particular tissue or organ. This suggests that thesediseases can be treated, and, furthermore, proper function of theaffected tissues and organs can be restored by targeting andmanipulating cell and tissue differentiation.

[0047] This work described herein, demonstrating the role of NO in cellproliferation and differentiation in an organism, provides for varioustherapeutic applications in humans and other mammals. In particular,this NO-based approach can be focused on renewable and regeneratingtissues, such as blood, bone, skin, and idgestive epithelium.Additionally, a similar strategy can be used to repopulate organs withnormally nondividing cells such as muscle and nerve cells.

[0048] The work described herein can also be used to enhance genetherapy methods. For example, NOS can be used to dirve a population ofcells in the S phase wherein the cells are replicating. As known in theart, replicating cells are more responsive to gene therapy methods(e.g., introduction of genes via live vectors) that non-replicatingcells. Thus, the present invention provides for a method of convertingcells into a state which renders the cells more receptive to genetherapy methods, wherein the cells are contacted with a NO inhibitor(e.g., NOS inhibitor). Conversely, the present invention provides for amethod of converting cells into a state which renders the cellsresistant to gene therapy methods. That is, the present inventionprovices for a method of converting cells into a state which renders thecells more resistant to gene therapy methods, wherein the cells arecontacted with NO and/or a NO enhancer (e.g., NOS enhancer).

[0049] The results of work described herein support the ability of NO toact as a crucial regulator of hematopoiesis after bone marrowtransplantation (BMT). NO regulates maturation of both erythroid andmyeloid cell lineages. By interfering with NO production in therecipient animal after BMT, the number of undifferentiated stem andblast cells which are the capable of further differentiation along theerythroid or myeloid lineages can be dramatically increased. The blastcells enrichment reaches 80-fold for the myeloid lineage, and 20-foldfor the erythroid lineage. The data described herein demonstrates thatmanipulations of NOS activity and NO levels during hematopoiesis can beused for therapeutic purposes to influence self renewal anddifferentiation of hematopoietic stem cells, and to replace damaged ordefective cells. Areas of application include enhancement of blood celland myeloid cell formation following high dose chemotherapy in cancertreatement; improved engraftment following bone marrow or stem celltransplantations, and gene therapy; stem cell therapy by amplifying theundifferentiated cells of erythroid and myeloid lineages and applyingappropriate factors to induce terminal differentiation; and regulationof formation of various blood cell components for treating hematologicaland autoimmune disorders.

[0050] the data also shows that changing the levels of NO productioninterferes with osteoblast and chondrocyte differentiation. Theseresults show that manipulation of NO production can regulate growth anddifferentiation of osteoblasts, chondrocytes, or mesenchymal stem cellsthis can be used for amplification and further differentiation of cellsin the injured tissue, or for cell implants (in combination withbiocompatible carriers, if necessary).

[0051] Thus, an NO-based approach can be used for regeneration therapyof the damaged tissue, post injury repair, age related diseases such asosteoporosis and osteoarthritis, and for reconstituting marrow stromafollowing high dose cancer chemotherapy.

[0052] In addition, the data shows that changing the levels of NOproduction interferes with keratinocytc differentiation the resultsdescribed herein demonstrate that regulation of NO production cam beused when increased proliferation and subsequent differentiation of skintissue is required (e.g., during burns and wound healing). Furthermore,NO can be used to control disorders caused by hyperproliferation ofkeratinocytes during psoriasis. Yet another potential application is touse NO-based preparations as exfoliant agents in cosmetic therapy.

[0053] NO has been shown to act as a regulator of cell differentiationin neuronal cells. It has been demonstrated that NO regulates braindevelopment in animals and contributes to controlling the size of thebrain in intact animals.

[0054] It has also been demonstrated that in certain contexts NOmediates the survival effects of growth factors by activating anantiapoptitic program and can protect neutonal cells from death.Combined, these studies of the role of NO in neurons suggest that NO maybe used to control proliferation and subsequent differentiation of nervecells in replacement therapy after neurodegenerative disorders caused byaging (e.g., Alzheimer's or Parkinson's), stroke, or trauma.

[0055] NO is actively produced in smooth muscle cells of the bloodvessels and is subject to complex physiological regulation. These cellsare highly susceptible to suppression of DNA synthesis by NO. The verystrong antiproliferative activity of NO can be used for inhibition ofsmooth muscle cells proliferation and neointima formation for treatmentof restenosis following angioplasty.

[0056] In addition, NO-based therapy has application for treatment ofailments characterized by destruction of specific sets of cells. Thisincludes heptanoate regeneration after toxic injury of the liver,treatment of reproductive system disorders, and administration ofdifferentiated pancreatic tissue for treatment of type 1 diabetes.

[0057] The methods of the present invention can be carried out in vivoor ex vivo. Administration of the NO inhibitor, NO enhancer and/or agentwhich induces differentiation can performed using various deliverysystems known in the art. The routes of administration include intradermal, transdermal, intramuscular, intraperitoneal, intravenous,subcutaneous, oral, epidermal and intranasally routes. Any otherconvenient route of administration can be used such as, for example,infusion or bolus injection, or absorption through epithelial ormucocutancous linings. In addition, the NO inhibitor, NO enhancer and/oragent which induces differentiation can be administered with othercomponents or biologically active agents, such as adjuvants,pharmaceutically acceptable surfactants, excipients, carriers diluentsand vehicles. Administration can be systemic or local, e.g., directinjection at the site containing the cells to be targeted. In theembodiment, in which the NO inhibitor, NO enhancer and/or agent whichinduces differentiation are protein or peptides, they can beadministered by in vivo expression of genes or polynucleotides encodingsuch into a mammalian subject. Several expression systems, such as livevectors, are available commercially or can be reproduced according torecombinant DNA techniques for use in the present invention.

[0058] The amount of NO inhibitor, NO enhancer and/or agent which foruse in the present invention which will be effective in the treatment ofthe particular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration and the seriousness of the diseaseor disorder, and should be decided according to the judgement of thepractitioner and each patient's circumstances. For example, the amountof NO inhibitor(s) for use in the methods of the present invention canbe from about 1 mg/kg body weight to about 1000 mg/kg body weight, fromabout 5 mg/kg body weight to about 500 mg/kg body weight, and from about25 mg/kg body weight to about 100 mg/kg body weight. In one embodiment,about 25 mg/kg body weight to about 1000 mg/kg body weight L-NAME and/orabout 1 mg/kg body weight to about 100 mg/kg weight can be used in themethods of the present invention. In a particular embodiment, about 300mg/kg body weight L-NAME is used in combination with about 30 mg/kg bodyweight ETU in the methods of the present invention.

[0059] the following Examples are offered for the purpose ofillustrating the present invention and are not to be construed to limitthe scope of this invention. The teachings of all references citedherein are hereby incorporated herein by reference.

EXAMPLES Example 1 Nitric Oxide Regulates Cell Proliferation duringDrosophila Development

[0060] Drosophila Stocks

[0061]Drosophila melanogaster Oewfon r strain was used for most of theexperiments described. Transgenic GMR-P35 flies (3.5 and 2.1 alleles,Hay et al., Dev., 120:2121-2129 (1994)) sere a generous gift from B. Hayand G. M. Rubin. transgenic flies carrying mouse macrophage NOS (NOS2)gene under heat-shoch promoter (hs-mNOS20(2) and hs-m NOS 15(2) alleles)were generated by P-element mediated germline transformation. A 4100base pair NotI fragment from the plasmid CL-BS-mac-NOS containing theentire mouse macrophagte NOS gene (Lowenstein et al., Proc. Natl. Acad.Sci. USA, 89:6711-6715 (1992)) was cloned into the Notl site in the Pelement vector pP(CaSpeR-hs) (Thummel and Pirrotta, DrosophilaInformation Service, 71:150 (1992)), placing it under the control of theDrosophila hsp70 promoter. The construct was coinjected into embryos(Spradling., P element-mediated transformation, In Drosophila. Apractical approach, D. B. Roberts, ed. (Oxford: IRL Press) 60-73 (1986))with the helper P element phs-II-Δ2-3 (Misra and Rio, Cell, 62:269-284(1990)). A set of two independent, homozygous transformants wereestablished. Expression of the NOS2 transgene after heat-shock treatmentof larvae and adult flies was confirmed by diaphorase staining and byprotein and RNA analysis. In control experiments, identical regimens ofheat-shock treatment of nontransformed flies did not induce anyanatomical changes per se.

[0062] Histochemistry and Electron Microscopy

[0063] NADPH-diaphorase staining was performed as described by Dawson etal., Proc. Natl. Acd. Sci. USA, 88:7797-7801 (1991) and Hope et al.,Proc. Natl. Acad. Sci. USA, 88:2811-2814 (1991), with minormodifications. Fixation-insensitive NADPH-diaphorase staining reflectsactivity of various NOS isoforms in mammal and Drosophila. Imaginaldiscs were mounted in 80% glycerol and photographed in a Zeiss Axiophotmicroscope under Nomarski optics. cobalt-sulfide staining of the pupalretinae was carried out as described by Wolff and Read, Dev.,113:825-839 (1991). BrdU labeling to identify cells in S phase wasperformed essentially as described by Schubiger and Palka, Dev. Biol,1223:145-153 (1987) and by Baker and Rubin, Dev. Biol., 150:381-396(1992), with minor modifications. Imaginal discs were removed, rinsed,and incubated in Schneider's media in 50 μg/ml solution of BrdU for30-40 minutes at room temperature. They 3were fixed in 4% formaldehyde,treated with 1:1 mixture of heptane and formaldehyde, rinsed,depurinated by 1 M HCl, blocked by 1% sheep serum, and incubated withanti-BrdU antibodies (Beckton-Dickinson). After extensive washing, discswere incubated with fluorsceine-coupled anti-mouse secondary antibodies(Boehringer-Mannheim). After rinsing individual imaginal discs weredissected away, dehydrated in ethanol, and mounted in Vectashieldmounting media (Vector Laboratories). Scanning electron microscopy wasperformed at the SUNY stony Brook Microscopy Center essentially asdescribed by Kimmel et al., Genes Dev., 4:712-727 (1990). The number ofommatidia was determined both by analyzing series of scanning electronmicrographs and by analyzing adult heads under the blue fluorescentlight in a Ziess Axiophot microscope.

[0064] Microinjection of Larvae

[0065] For inhibition of NOS, third instar larvae were injected withL-nitroarginine methyl ester (L-NAME), its inactive enantiomerD-nitroarginine methy ester (D-NAME) (both from Sigma), and2-ethyl-2-thiopseudourea (ETU; Calbiochem). Chemicals were dissolved inSchneider's solution at concentrations of 0.1 M for L-NAME and D-NAMEand 0.01 M for ETU and mixed with Freund's adjuvant (Sigma) in 1:3ratio. Amounts of 5-10 nl were microinjectd in staged late third instarusing a glass needle. Timing of the injection of NOS inhibitors thatgave the highest efficiency (as determined by the changes in thephenotype of tha adults) was determined in trial experiments and wasfound to be most efficient whtn performed 5-12 hours before pupanation.This treatment did not affect the onset of pupanation and hatching.

[0066] Ectopic Expression of NOS

[0067] For regulated ectopic expression of NOS, larvae carrying themouse NOS2 cDNA under the control of Drosophila heat-shock promoter weretreated with heat shock at 36° C. for 40 minutes within the first hourafter puparium formation. For BrdU lableing experiments, third instasrlarvae were treated with heat shock 5-8 hours before puparium formation.

[0068] Results

[0069] NOS is expressed in imaginal discs during larval development.

[0070] At the end of the third instar, cells of imaginal discs undergotemporary cell cycle arrest. Cytostasis is released 12-14 hours afterpupariation and is established once again (permanetly) in the late pupaeand the pharate adult. The ability of NO to reversibly halt celldivision and establidh temporary growth arrest makes it a plausiblecandidate for mediating cytostasis in imaginal discs. To investigatethis possibility, imaginal discs of the third instar and early pupaewsere examined for NOS presence. Drosophila NOS (dNos) gene, which ispreferentially expressed in the adult head, has recently been cloned andcharacterized (Regulski and Tully, Proc. Natl. Acad. Sci. USA,92:9072-9076 (1995)). However, different NOS-related mRNA species arepresent in the embryo, larvae and adult flies. These mRNAs may beproduced by the cloned dNOS gene or by other potential Drosophila NOSgenes, making the detection of the relevent RNA species difficult.Therefore, to visualize the expression of NOS in Drosophila duringlarval development, histochemical staining for the NADPH-diaphorase(reduced nicotinamide adenine dinucleotide phosphate-diaphorase)activity of NOS was used, which reflects the distribution of the totalenzymen activity in a tissue (Dawson et al., Proc. Natl. Acad. Sci. USA,88:7797-7801 (1991); Hope et al., Proc. Natl. Acad. Sci. USA.88:2811-3814 (1991); Muller, Eur. J. Neurosci., 6:1362-1370 (1994)).

[0071] NADPH-diaphorase staining was observed in all imaginal discs,imaginal rings, histoblasts and the brain of the larvae, beginning inthe third instar. Staining became more intense as development proceeded,and in late third instar larvae and early pupae, a highly specific andreproducible pattern of very intense staining was evident. In the legimaginal disc NAPDH-diaphorase staining was intially seen at the verybeginning of the third instar. Staining was confined to the center ofthe disc, corresponding to the presumptive distal tip of the leg. As thediscs matured, diaphorase staining intensified, and in the late thirdinstar it nearly obliterated the distinction between individualconcentric rings of epithelial folding normally seen in axial view. Atthe end of the third instar stage, the staianing of the center of thedisc (distal tip), which stained most darkly at the beginning of thethird period, was weaker in comparsion with the surrounding cells. Laterin development, when the discs began to evert in the prepupae,diaphorase staining of the forming leg became less intense, and adistinct characteristic pattern of staining of individual segmentsbecame evident. At 2-4 hours after puparium formation, intenseNADPH-diaphorase staining was observed in the presumptive tibia, firstand second tarsal segments, and the proximal part of the fifth tarsalsegment of the forming leg. Staining was much weaker in the third andfourth segments, and areas of intense staining were unevenly distributedthroughout the regions of presumptive femur. Weak staining was alsopresent in the coxa and body wall. The progression of staining patterns.throughout the larval development was highly specific and reproducible.The staining of the imaginal discs corresponding to the first, secondand third pairs of legs was very similar. As with the leg imaginaldiscs, other imaginal discs, imaginal rings and histoblasts exhibitedincreasingly intense NADPH-diaphorase staining as larval developmentproceeded. Wing, eye, haltere and genital discs in the third instar haddistinct and reproducible patterns of intense staining, which graduallydecreased in a specific spatial pattern during early pupal development.

[0072] These results demonstrated that there is a gradual and specificaccumulation of NOS in developing imaginal discs, which reaches highestlevels at the time wnen the progression through the cell cycle slowsdown.

[0073] Synthesis of DNA is affected by manipulations of NOS activity

[0074] If NO acts as an antiproliferative agent during Drosophiladevelopment at stages when the cells of imaginal discs enter temporarycytostasis, them its action might directly affect DNA synthesis in thediscs. Inhibition of NOS would then be expected to relieve the block andincrease the number of cells in S-phase; conversely, high levels of NOwould lead to a decrease in the number of dividing cells. To test thishypothesis and to map the extent and distribution of theantiproliferative effect of NO, DNA synthesis in larval and prepupaldiscs was monitored while the levels of NOS activity were manipulated.To inhibit NOS activity, specific NOS inhibitors were injected intodeveloping larvae. To increase the levels of NOS, expression of NOStransgene was induced in transformed larvae carrying the mouse NOS2 cDNAgene (Lowerstein et al., Proc. Natl. Acad. Sci. USA, 89:6711-6715(1992)) under the control of the heat shock promoter NOS2 is acalcium-independent form of NOS that is capable of efficientconstitutive NO production. Imaginal discs were labelled with5-bromo-deoxyuridine (BrdU), and the extent and distribution of labelingof S-phase nuclei in leg imaginal discs from larvae after inhibition ofNOS, from NOS2 transformants after heat shock induction, and fromcontrol untreated larvae were compared. The data show that there weresignificantly more BrdU-labled cells in imaginal discs of larvae inwhich NOS activity was supressed by L-nitroarginine methyl ester(L-NAME) than in control untreated larvae (or larvae treated with theinactive isomer D-NAME). The data also show that there weresignificantly more BrdU-labled cells in imaginal discs of flies in whichNOS was inhibited than in control flies. In contrast, there weremarkedly fewer BrdU-labeled cells in imaginal discs from inducedNOS-transformed flies than in uninduce controls. At the same time, thesechanges in the number of BrdU-labeled cells after inhibition or ectopicexpression of NOS appeared to be evenly distributed over the entiredisc.

[0075] These data indicate that modualtion of NOS activity affects thenumber of cells in S phase in imaginal discs, which is consistent withthe observations that NO suppresses DNA synthesis and cell division.

[0076] Inhibition of NOS results in hypertrophy of leg segments

[0077] The highest levels of diaphorase staining occur during the periodof development when DNA synthesis and the rate of cell division in mostof the imaginal disc cells slow down. The strong antiproliferativeproperties of NO and the specific pattern of diaphorase staining seen inmature imaginal discs impled that NO might act as a growth arrest agentin these structures, capable of inhibiting DNA systhesis and supportingtemporary cytostasis during the switch to metamorphosis. If NO indeedacts as an anti-proliferative agent during the late stages of larvaldevelopment, then inhibition of NOS might result in excessive growth oforgans and tissues, whereas ectopic overexpression of the NOS gene mighthave the oppositie effect.

[0078] To test this hypothesis, NOS activity was inhibited by injectingspecific NOS inhibitors in the developing larvae at the end of the thirdinstar, several hours before metamorphosis. Most of the larvae completedmetamorphosis successfully, giving rise to adult flies within the normaltime frame. The resulting adults differed from normal flies in manyrespects, the most dramatic being enlargements of the appendages andother structures of the fly body. The changes included a) hypertrophy ofthe demur, tibia and the segments of the tarsus; b) overgrowth of thetissues orginating from the genital discs; in extreme cases, these cellscontributed to more than one-quarter of the fly body; c) and increase ofthe overall surface of the wings; d) overgrowth of the cells of tergitesand sternites; e) hypertrophy of the humerus; f) occassionalduplications of some areas of the eye; g) occasional realformation ofgenital structures, legs and eyes; and h) occasional ectopic formationof misplaced body structures.

[0079] The changes were most profound in, and most often affected thelegs of, the adults. The hypertrophy was particularly strong in thethird pair of legs, where the diameter of certain segments increased 3-4fold. The number of bristles and the number of rows or bristles alsoincreased, confirming that hyperproliferation of the cells had occurred.The leg segments most strongly affected were those (first and secondtarsa segments, tibia, and femur) whose primordia had the highest levelsof NOS at the larval and prepupal stages. The changes affected mainlythe anteroposterior and dorsoventral but not the proximodistal axes, sothat the length of the affected segments remained the same. Identicalchanges were observed when two structurally unrelated inhibitors of NOS,2-ethyl-2-thiopseudourea (ETU) and L-NAME (But not D-NAME) were used,indicating that the observed effect resulted specifically from blockingNOS activity.

[0080] In summary, these data show that inhibition of NOS at the latestages of larval development results in excessive cell proliferation andincreased size of the structures of the body of the adult fly.

[0081] Ectopic expression of a mous NOS transgene results in reducedsize of leg segments

[0082] The ability of NO to inhibit DNA synthesis and cell proliferationsuggests that overexpression of NOS in developing larvae may lead todiminished cell proliferation in the imaginal discs and to a reductionin the size of organs of the adult fly. Transformed flies that expressthe mouse NOS2 transgrene under the control of the heat-shock promoterwere tested. Transgenic larvae were heat-shocked within one hour afterpupariation to inbduce etopic expression of NOS before the final celldivisions take place. This resulted in a reduction in the size of thelimbs of the fly. The distal segments of the legs were affected mostfrequently and to the greatest degree. In extreme cases, the wholetarsus was shortened 1.5-2 fold, and the third, fourth and fifthsegments were fused together with poorly defined boundaries. The numberof bristles in a row on the affected segments also decreased, althoughthe number of rows did not change. The segments of the adult leg mostoften affected by the overexpression of NOS (third, fourth and fifthtarsal segments) were those that were not affected by the NOS inhibitorsand whose precursors exhibited particularly low levels of diaphorasestaining in the early prepupal stages. The most terminal structures ofthe appendage, including the tarsal claw, remained intact in thesedefective legs. This suggests that the observed reduction in size wasdue to incomplete growth of the distal area of the developing appendage,rather than to complete loss of its distal structures. In contrast tothe results on NOS inhibition, the changes affected only theproximodistal axis, while the diameter of the affected segments remainedthe same. In addition to the reduction in the size of the leg segments,changes included a decrease in the overall surface of the wings, cuts inthe wings, and reduced size of tergites and sternites.

[0083] The results support the conclusion that ectopic expression of NOSat the late stages of larval development results in a decrease in cellproliferation and a reduction in the size of the structures of the bodyof the adult fly.

[0084] Inhibition of Apoptosis Unmasks Excessive Proliferation

[0085] In leg imaginal discs, the changes in the number of S-phasenuclei after manipulation of NOS activity directly correlated with thechanges in the size of the adult limbs. However, in the eye imaginaldisc, an increase in the number of cells in S-phase was consistentlydetected after inhibition of NOS, but the resulting adult eye usuallyappeared normal. The possibility that the apparently normal eyephenotype occured as a result of programmed cell death, whichcounteracts excessive cell proliferation induced by NOS inhibition andrestores the normal number of cells in the eye during metamorphosis, wastested. To suppress programmed cell death, GMR-P35 flies were used (Hayet al., Dev., 120:2121-2129 (1994); donated by Drs. B. Hay and G. Rubin)in which apoptosis in the developing eye is largely prevented byexpression of recombinant baculovirus p35 protein. p35 is a stronginhibitor of aproptosis, which acts by inhibiting the interleukin1B-converting enzyme-like proteases and is able to prevent aproptosis inmultiple contexts. GMR-P35 flies express p35 under the transcriptionalcontrol of multimerized glass-binding site from the Drosophila Rhlpromoter. Glass promoter directs expression of the transgene in allcells in and posterior to the morphogenetic. furrow in the eye disc(Ellis et al., Dev., 119:855-865 (1993)).

[0086] When NOS was inhibited in GMR-P35 larvae, the eyes of the adultflies showed numerous changes, reflecting the excessive proliferation ofvarious cell types in the developing eye. The most dramatic of thesechanges was in the number of ommatidia in the adult eye, which increasedfro the nearly invariant complement of 750 in wild type flies (747+/−4)and untreated GMR-P35 flies (748+/−6), to nearly 820 (818+/−21) afterNOS inhibition in GMR-P35 flies. This, together with the elevated numberof cells per ommatidium, caused an increase in the overall size of theeye. Other changes in p35-expressing flies after inhibition of NOScompared with the control GMR-P35 flies included a) more ommatidia withan irregular shape (perhaps, because of the uneven increase in thenumber of various cell types); b) more ommatidia with an irregulararrangement of the rows; and c) more ommatidia of a smaller size.

[0087] Another manifestation of the inhibition of NO production inGMR-P35 flies was an in crease in the number of pigment, cone, andbristle cells. Wild type ommatidia contain, in addition to eightphotoreceptor cells, a set of four cone cells and two primary pigmentcells, surrounded by an array of six secondary pigment cells, thereetertiary pigment cells, and three bristles (Wolff and Ready, Patternformation in the Drosphila retina, in the Development of Drosophilamelanogaster, M. Bate and A. Martinez-Arias, eds. (Cold Spring HarbortLaboratory Press, cold Spring Harbor, N.Y.), 1277-1326 (1993)). Thenumber of photoreceptor and accessory cells is normally constant, andvariations in this arrangement in the eyes of the normal flies are veryrare. In GMR-P35 flies, the number of secondary and tertiary pigmentcells was increased from 12 to 25 (25+/−4) cells per sample area(defined as described in Hay et al., Cell, 83:1253-1262 (1995)) as aresult of suppressed programmed cell death. Inhibition of NOS in theflies resulted in a further increase in the number of secondary andtertiary pigment cells to more thant 35 (36+/−8) per sample area. Thisnumber exceeds the maximal number of pigment cells saved from programmedcell death in untreated GMR-P35 flies and suggests that extra pigmentcells arise as a result of excessive cell proliferation caused byinhibition of NOS combined with suppression of cell death caused by p35.

[0088] The number of ommatidia with extra primary pigment cells inGMR-P-b 35 flies after inhibition of NOS was also increased incomparison with control flies, although it only slightly exceeded thelevels in untreated GMR-P35 flies after NOS inhibition, up to 1.5 perommatidum instead of the three seen in normal flies and untreatedGMR-P35 flies, and these were often mislocated. Similarly, the number ofcone cells was increased from four in normal and untreated GMR-P35ommatidia to five and six in many ommatidia of GMR-P35 flies after NOSinhibition. Clusters of ommatidia were also found which contained one,two, or three cone cells, which may correspond to improperly formedsupernumerary ommatidia that did not attain the proper set of cells.

[0089] Thus, prevention of apoptosis by baculovirus p35 protein in thedeveloping eyes of transgenic flies evealed excessive proliferation ofvarious cell types after NOS inhibition in larvae, which was otherwisemassked by programmed cell death in the larvae and pupae.

EXAMPLE 2 Nitric Oxide Regulates Hematopoiesis in Animals ErythroidDifferentiation

[0090] To study the formation of cell of the erythroid lineage in thespleen of the irradiated recipient mice, the animals (females of FlCBA×C57B1 hybrids weighing 22-24 g) were treated with 750 cGy total bodyirradiation within 3-4 hours before transplantation. This dosage wastested to be enought for complete suppression of hematopoiesis in theirradiated recipient animals. BM cells were flushed from the femurs ofsyngeneic donors and injected intravenoulsy (10⁵ BM cells per mice) inthe recipients. The animals received twice a day injections of 100 mg/kgof L-NAME and D-NAME and 10 mg/kg of ETU for 7-10 days. Mice wereanalyzed 9-10 days after transplantation.

[0091] The differentiation status of the colonies in the spleen wasevaluated by morphological criteria and by immunohistochemical tests forthe presence of receptors to various cytokines, which are present onlyat specific stages of the erythroid cells' treated with inactiveenantiomere D-NAME (Table 1) showend that in agreement withnumerous-data, most of the colonies in the spleen (>60%) containederthroid colonies, smaller fractions contained undifferentiated blastscells (14%) or both erthroid and blast cells (13%), and small fractionsof colonies contained megakaryocytes (7.5%) and granulocytes (4%). Incontrast, when animals were treated with NOS inhibitors after BMtransplantation, most of the colonies in the spleen containedundifferentiated blast cells (up to 85% of blast cells colonies andmixed blast crythroid cells colonies). Erythroid colonies comprised only15% of the total number of colonies, and the megakaryocyte andgranulocyte colonies were not detectable. The results were similar withtwo structurally unrelated NOS inhibitors, confirming the specificity oftheir action. Thus, prolonged treatment of recipient mice after BMtransfer with NOS inhibitor, reversed the ratio of blastcells-containing colonies to the erythroid cells-containing coloniesalmost 16 fold, effectively preventing erythroid differentiation. TABLE1 Formation of hemopoietic colonies in spleens of irradiated mice afterinjection of NOS inhibitors. blast cell and blast erythroid megakarycell cells erythroid ocyte granulocyte colonies colonies coloniescolonies colonies Experiment 54.6% 30.25% 15.12% — — Control 14.3% 12.9%61.22% 7.48% 4.08%

[0092] Myeloid Differentiation

[0093] To study the formation of cells of the myeloid lineage, celluloseacetate membranes were implanted in the peritoneal cavity of mice. After7 days, when a layer of fibroblasts had covered the membranes, the micewere irradiated as described above. BM cells from syngeneic donors wereinjected (10⁵ BM cells per mice) in the peritoneal cavity of therecipients. Animals received injections of NOS inhibitors as describedabove. Membranes with growing colonies were isolated and analyzed 7-8days later.

[0094] The differentiation status of the colonies in the spleen wasevaluated by morphological criteria, myeloperoxidase rreaction, and byimmunohistochemical tests for the presence of receptors to variouscytokines, which are present only at specific stages of the myeloidcells' maturation. The analysis of the colonies on the membranes incontrol animals and animals treated with inactive enantiomere D-NAME(Table 2) showed that in agreement with numerous data, most of thecolonies (92%) contained granulocytic colonies. A much smaller fractioncontained undifferentiated blasts cells (6%), and a very small fractionof colonies contained erythroid cells (1.3%). In contrast, when animalswere treated with NOS inhibitors after BM transplantation, most of thecolonies on the membranes (up to 85%) contained and undifferentiatedblasts cells. Colonies with differentiated cells of the granulocytelineage comprised only 15.6% of the total number of colonies, and anegligible fraction of the colonies (<0.5%) contained erythroid cells.The results were similar with two structurally unrelated NOS inhibitors,confirming the specificity of their action. Thus, prolonged treatment ofrecipient mice after BM transfer with NOS inhibitor, reversed the ratioof blast cells-contiaing colonies to the granulocytic colinies almost80-fold, effectively preventing myeloid differentiation. TABLE 2Formation of hemopoietic colonies on cellulose acetate in the peritonealcavity of irradiated mice after injection of NOS inhibitors.granulocytic blast cell colonies colonies erythroid colonies Experiment84.46% 15.6% — Control  6.34% 92.3% 1.34%

[0095] Differentiation Status of Transplanted BM Cells

[0096] To study the stage to which the transplanted cells haveprogressed, colonies in the spleen and on the membranes were tested withspecific antibodies for receptors of various growth factors. Thisanalysis permits one to visualize and evaluate the stage of themultistep differentiation process that eventually leads to erythroid ormyeloid differentiation. we have used antibodies specific from thereceptors to interleukin 3(IL-3-R), granulocyte macrophage colonystimulting factor (GM-CSF-R), granulocyte colony stimulating factor(G-CSF-R) and erythropoietin (EpoR). The appearance of each or thesereceptors marks a specific stage in hematopoiesis.

[0097] The results of the analysis demonstrate that the blast cells inthe spleen colonies (representing erythroid differentiation) haveaccumulated mostly at the stage of differentiation where they havealready acquired the receptor for IL-3, but not for erythropoietin,GM-CSF or G-CSF, whereas the colonies with morphological signs oferthroid differentiation had accumulated EpoR.

[0098] the blast cells in the colonies on the membranes (representingmyeloid differentiation) have accumulated mostly at the state ofdifferentiation where they have alread acquired the receptor for IL-3,but not for erythropoietin, GM-CSF or G-CSF, whereas themyeloperoxidase-positive colonies with morphological signs of myeloiddifferentiation had accumulated GM-CSF-R and G-CSF-R.

[0099] Stem Cells in the Bone Marrow

[0100] To study the maturation of hematopoietic cells in the bone marrowof the irradiated recipient mice, the animals were treated as describedabove and the BM cells from the demurs of syngeneic donors were injectedintravenoulsy (10⁵ BM cells per mice) in the recipients. The animalsreceived injections of NOS inhibitors (L-NAME, its inactive enantimereD-NAME and ETU) as described above, and mice were analyzed 7-10 daysafter transplantation.

[0101] The BM cells were tested fro the presence of various growthfactor receptors which serve as markers of the differentiation stage andindicate the presence of stem cells and multipotent percursor cells. TheBM preparations were tested for cells expressing receptors to HSF(ligand of c-kit), GM-CSF, G-CSF and IL-3. The results Table 3 show thatinhibition of NO synthesis in the recipient animals after BM transferleads to dramatic increase in the number of c-kit positive andIL-3-R-positive cells, siggesting that the pupulation of cells in the BMbecomes highly enriched in hematopoietic stem cells. At the same timethe number of cells expressing receptors for G-CSF, which marks thelater stages of differentiation, decreses almost three-fold, while thenumber of GM-CSF-R-positive cells is slightly decreased. This suggeststhat inhibition of NOS during hematopoiesis selectively enriches the BMin undifferentiated stem cells which have already acquired c-kit and IL3receptors, but have not proceeded to the later stages when the receptorfor G-CSF is synthesized. TABLE 3 Presence of hematopoietic markers inBM cells of irradiated mice after injection of NOS inhibitors Markersc-kit IL-3-R G-CSF-R GM-CSF-R control (no injections)  2%  7% 24% 18%treatment with L-NAME 46% 58% 12% 13% treatment with ETU 84% 83%  8% 10%treatment with D-Name  7% 12% 19% 16%

[0102] Reversibility of the NOS inhibitors' Action in BM cells

[0103] The critical question is whether undifferentiated stem cellswhich accumulate in the bone marrow as a result of treatment with NOSinhibitors have the capacity of revert to normal state and resume normalhematopoiesis process once the action of NOS inhibitors is suspended.The failure to do so might indicate that the cells become stranded intheir undifferentiated status, similar to various pathologicalconditions to answer this question, the treatment of mice with NOSinhibitors was halted 7-9 days after the BM transfer and checked the BMcells for the presence of hematopoiesis markers 1-7 days aftertermination of injections. Control mice continued to receive the dailyinjections, the results (Table 4) demonstrate that once the treatmentwith inhibitors of NOS is suspended, the cells were able to resume theirdifferentiation and to proceed to the later stages normally. Thisindicates that enrichment in stem cells after treatment with NOSinhibitors is reversible and can be used to “bost” the number of stemcells before inducing the to proceed further along their differentiationpathways. TABLE 4 Presence of hematopoietic markers in BM cells ofirradiated mice after injection of NOS inhibitors and subsequentsuspension of treatment Markers c-kit IL-3-R G-CSF-R GM-CSF-R treatmentwith L-NAME for 78% 77%  9% 11% 8 days treatment with L-NAME for 82% 84% 9% 12% 13 days 1 day after suspension of 62% 71% 15% 14% treatment 2days after suspension of 29% 37% 16% 14% treatment 3 days aftersuspension of  9% 28% 18% 18% treatment 5 days after suspension of  8%14% 28% 21% treatment 7 days after suspension of  5% 12% 26% 20%treatment

[0104] NOS Inhibition and Apoptosis

[0105] To test whether prolonged treatment with NOS inhibitors affectsthe rate of programmed cell death in BM cells, the number of aproptoticcells in the preparation of BM cells was examined. The TUNEL approachwas used, thus revealing the cells with intensely fragmented DNA, ahallmark of apoptosis, at the same time using the DAPI staining tovisualize the nuclei of all cells in the preparation. The resultsindicate that neither prolonged treatment with L-NAME, nor with ETU didnot affect the porportion of apoptotic cells (8+/−3% in control versus7+/−in L-NAME treated and 8% +/− in ETU-treated animals). Similarly,suspension of treatment with inhibitors did not affect programmed celldeath in BM preparations (9+/−45 of TUNEL-positive cells). This suggeststhat manipulation of NOS activity in the animals after BMT, althoughhaving profound effect on differentiation and maturation ofhematopoietic cells, does not affect the extent of programmed cell deathin BM cells, further supporting the feasibility of applications of NOSinhibitors for therapy.

EXAMPLE 3 Nitric Oxide Regulates Brain Development in Vertebrates

[0106] It has been recently demonstrated that nitric oxide (NO), amultifunctional second messenger, is involved in cell and tissuedifferentiation and organism development. NO synthase (NOS) controls thetransition from cell proliferation to growth arrest and, as a result,regulates the balance between cell proliferation and differentiation incultured neuronal cells, in developing Drosophila, and duringhematopoiesis in mammals (Peunova et al., 1996; Kuzin et al., 1996;Michurina et al., 1997). Here, whether NOS is involved in the braindevelopment in vetebrate animals was tested. Xenopus laevis was chosenas a model organism for these strudies, focusing the investigation onthe formation of the brain. The Xenopus NOS gene was cloned and thedistribution of NOS-positive neurons in the developing brain wasstudied. It was found that inhibition of NOS dramitically increases thenumber of cells in the developing brain, and increases the overall sizeof the brain. The results suggest that NOS is directly involved in thecontrol of cell proliferation and neuronal differentiation in thedeveloping vertebrate brain.

[0107] Cloning of the Xenopus NOS Gene

[0108] Using the information about the known NOS genes, the NOS cDNAfrom Xenopus (XnNOS) was cloned. Analysis of its primary structurwesuggests taht the cloned gene represents the homologue of the Ca2⁺-dependent neuronal NOS isoform of mammals. Analysis of the genereveals a remarkable degree of evolutionaly conservation with longstretches of amino acid sequences identical to those of humans, mice,rats, and Drosophila. The cloned gene produces enzymatically activeprotein when transfected in cultured cells. The primary structure of thegene made it possible to obtain a specific antibody, and theimmunofluorescence analysis indicates that the diaphorase staining ofthe developing Xenopus correctly represents the distribution of theXnNOS enzyme this notion is supported by in situ hybridization analysisof XnNOS transcripts in the tadpole brain. The cloned gene is now beingused to isolate other putative NOS genes from Xenopus.

[0109] NOS is Expressed in a Consistent Spatio-Temporal Pattern in theDeveloping Xenopus Brain

[0110] The Xenopus brain undergoes histogenesis starting at stage 39-40;prior to that, the neural tube consists of rapidly dividingundifferentiated neuroepithelial cells. In the growing brain of theXenopus tadpole, new cells arise in the narrow zone of the germinallayer in a defined pattern, which can be revealed by labeling with BrdR.The distribution of NADPHY-diaphorase staining (which is indicative ofNOS expression) in Xenopus brain from stage 40 through stage 50 wasanalyzed. Zones of staining first appeared at stage 43, the time ofmigration of young neurons off the neural tube and theirdifferentiation. Staining appeared outside of the germinal layer andbecame more intense as development of tadpoles proceeded. The mostintense staining was observed in single large differentiated neurons inthe tectum and spinal cord, and in the marginal zone of the tectumcomposed of processes of differentiated neurons. The gradient ofdiaphorase staining was latero-medial and reciprocal to the pattern ofproliferation, suggesting that zones of active proliferation in thegerminal layer remained free of NOS activity through these stages.Inhibition of NOS in the developing brain resulted in excessiveproliferation of young neurons.

[0111] To test whether NOS is involved in growth arrest in neuronalprecursors in the developing Xenopus brain, NO production was blocikedby introducing pieces of plastic impregnated with NOS inhibitors, L-NAMEand ETU, into the ventricle of the tadpole's brain at state 43. After 3,7 and 12 days, animals were examined for changes in the patterns of celldivision, differentiation, survival, and morphology of the brain. BrdUlabeling demonstrated a dramatic increase in the number of cells in theS phase of the cell cycle in the inhibitor treated brains, compared tothe control brains. The number of BrdU-positive cells in the tectumconsistently increased throughout the experiment. Staining of cellnuclei with DAPI revealed higher number of cells in the brain sectionsin each time interval of the experiment, indicating that excessive cellsin the S phase successfully completed the cell cycly by mitosis.

[0112] Inhibition of NOS and Programmed Cell Death

[0113] Whether the inhibition of NOS and excessive proliferation ofcells in the developing brain affects the programmed death of neurons inthe tectum was tested. Using the TUNEL technique to visualize theapoptotic cells in the brain, it was found that at day 3 the number ofTUNEL-positive cells was the same in both the control and inhibitortreated tectum. However, after 7 and 12 days, there were more apoptoticcells in the brains of animals which received NOS inhibitors, than incontrol animals. The increase in the number of TUNEL-positive cells isnot due to toxicity of the inhibitors, since cells continued toincorporate BrdU very effectively. Identical changes were obserces withtwo structurally unrelated inhibitors of NOS, indicating that theeffects resulted specifically from blocking NOS activity. This datasuggests that excessive proliferation of cells in the tectum leads toactivation of programmed death acting to remove the surplus neurons.Alternatively, this may indicate that differentiated neurons becamedependent on NO for survival, similar to the situation i nfulldifferentiated PC12 cells (Peunova et al., 1996). Neuronaldifferentiation in the brain is affected

[0114] to test whether excessive cell proliferation induced by NOSinhibitors affects the distribution and differentiation of neurons inthe Xenopyus brain, antibodies to specific neuronal markers which have aspecific and highly reproducible pattern of expression during Xenopusdevelopment were used. It was found that distribution of neuronspositive for Islet-1, N-tublulin, and N CAM was changed after inhibitionof NOS. In particular, the neurons were sisplaced into the marginalzone, neurons in the intermediate layer were more heterogeneous and withshorter branches than in control brains, and the distinct layeredstructure of the tectum was altered. In addition, the number of Islet-1positive motor neurons was increased after inhibition of NOS. Inhibitionof NOS leads to ectopic proliferation of neuronal precursors.

[0115] The Xenopus brain has a fine cytoarchitecture. Groups ofneighboting cells share the place and time of birth and become involvedin common local circuits. The position of young and mature neurons inthe brain is strictly dependent on the place of their birth, migration,and final differentiation, and compose a characteristic pattern. In thebrains of animals treated with inibitors of NOS, it was found, inaddition to extra layers of young dividing neuronal precursors, numerousectopic sites of neuronal proliferation. Large clusters of cells wereobserved in atypical location, occupying the marginal zone, variousareas of the tectum, the telencephalon and the hindbrain.

[0116] Inhibition of NOS Increases the Overall Size of the Brain

[0117] Inhibition of NOS activity in the brains of developing tadpolesresulted in increased number of cells in the S phase, accompanied by amodest increase in programmed cell death at late stages. Together, thisincreased the total number of cells in the brain and consequentlyincreased the overall size of the brain. The most affected areas are theoptic tectum and the area immediately adjacent to the ventricle wherethe impregnated piece of plastic was inserted. In cases when the sourceof the NOS inhibitor was shifted in the ventricle towards thetelencephalon or hindbrain regions in the developing brain an increasein size of the anterior the posterior parts of the brain, respectivelywas observed.

[0118] Taken together, these results demonstrate that NO controls thenumber of neurons in the developing brain, and inhibition of NOSdirectly affects the size of the Xenopus brain. This confirms the roleof NO as a general regulator of cell and tissue differentiation in theorganism. This suggests that manipulations of the NO levels may be usedfor therapeutic purposes to control proliferation and subsequentdifferntiation of nerve cells in replacement therapy afterneurodegenerative disorders caused by aging (e.g., Alzheimers,Parkinson's or Huntington's), stroke, or trauma.

EXAMPLE 4 Nitric Oxide and Hematopoietic Stem Cell Enrichment Materialsand Methods

[0119] Animals

[0120] Female mice were used at 8-12 week of age, and were of thefollowing strains: C57B1/6, B6 CBAF1/J, CBAB6F1/J, DBA (purchased fromJackson Laboratories or Taconic Farms). All mice were bred andmaintained at the Animal Care facility of CSHL on standard food diet andacidified water ad libidium.

[0121] Irradiation and Bone Marrow Transplantation

[0122] Recipient mice were exposed to 8.2-9.5 Gy total body gammairradiation using Marc I irradiator from Cesium-137 source (AtomicEnergy of Canada, Ottawa) at a dose rate of 1.06 Gy/min 3-20 hoursbefore bone marrow transplantation. The dose of irradiation is enough tosuppress hematopoiesis in recipient mice. NO action on hematopoiesis wasstudied by BM transfer after total body irradiation. The donor mice weredacrificed by CO2 asphyxiation or cervicl dislocation and the femuresand tibiae were isolated. The bone marrow cells were extracted from thefemures and tibiae were isolated. The bone marrow cells were extractedfrom the femures and tibiae by repeatedly flushing the bones withDulbecco modified Eagle medium (DMEM) (GibcoBRL). Single cellsuspensions were prepared by drawing the bone marrow through a 21-guageneedle folowed by a 26-guate needle and through 70 mkm nylon cellstrainer. Cells were counted using a hematocytomerer. 3-5×10⁴ nuclearbone marrow cells were injected into tail vein or 1×10⁶ cells wereinjected intraperitoneally. Spleens or testis were cut into pieces, thendraw through a 21-guage needle and a 70 mkm nylon cell strainer toobtain a single cell suspension.

[0123] Spleen Colony Assay

[0124] The spleen colony assay of Till and McClulloch (Till, J. E. andMcCulloch, E. A., Radiat. Res., 14:213 (1961)) was applies. 3×10⁴ bonemarrow cells were injected into lethally irradiated mice (8.5-9.5 Gytotal body irradiation from a Cesium-137 source at a dose a 1.06Gy/min). The spleens were removed on days 8 or 12 after transplantation,fixed in Carnua's (96% ethanol: chlorophorm: acetic acid: 6:3:1) orBouin's solution (sigma), and macroscopically visible spleen colonieswere counted. Secondary transfer of bone marrow or spleen cellsuspensions were assayed 12 days after primary transplantation. Thenumber of day-8 and day-12 spleen colonies in secondary animals wascounted.

[0125] NOS Inhibitor Administration

[0126] N-omega-Nitro L-arginine (L-NAME) (Sigma), N-omega-NitroD-arginin (D-NAME) (Sigma) and 2-ethyl-2-thiopseudourea hydrobromide(Calbiochem) (ETU) were used. To suppress NOS activity in the recipientanimals, they were injected intravenously or intraperitoneally with 0.3ml of a mixture of two NOS inhibitors L-nitro-methylester (L-NAME) at300 mg/kg of body weight, and 2ethyl-2-thiopsudourea (ETU) at 30 mg/kgof body weight immediately after bone marrow transplantation. Suchinjections were repeated twice a day for 3-17 days. In differentexperimental groups of animals the treatment was suspended after 3, 5, 7or 9 days. The animals in the control group received injections of 0.3ml of saline solution.

[0127] FACS

[0128] to prepare cells for FACS, mice were killed by cervicaldislocation and bone marrow cells from both femurs and tibiae wereflushed out using a 2 mL syringe with 21-gauge needle followed by26-guage needle. Spleens and testis were cut into pieces, then drawnthrough a 21-guage needle and a 70 mkm nylon cell strainer to obtain asingle cell suspension. Bone marrow, spleen, or testis cells werecounted using a hematocytometer,. After wasing in MEM (Minimal EssentialMedium, GibcoBRL) and was solution 3% fetal bovine serum on PBS)hemapoietic cells (bone marrow or spleen cells) were resuspended in PBS(phosphate buffered saline) containing 3% fetal bovine serum.Erythrocytes were lysed with ammonium chloride-potassium bicarbonatebuffer (154 mM ammonium dichloride, 10 mM potassium bicarbonate, 0.082EDTA) 5 minutes at room temperature. After washing, cell suspensionswere filtered through a 70 mkm pore size nylon cell strainer and werecounted using a hemacytometer. 3 5×10⁶ nuclear hemopoietic cells in 50ul PBS supplemented with 3% fetal bovine serum were incubated with 50 ulantibodies for 20-40 minutes at 4+ C. in the dark. Then cells werewashed twice with PBS and fixed with 300-500 ul of 2% formaldehyde inPBS. The negative controls were unstained cells or cells stained withonly second antibodies. All cells were kept on ice throughout the wholeprocedure. Fixed cells were kept in the refrigerator at 4° C. till flowcytometry analysis. Control and stained samples were analyzed using anEPICS Elite cell sorter (Coutler, Hialeah, Fla.).

[0129] Antibodies

[0130] The antibodies used in immunofluorescence staining includedE13-161.7 (anti-SCA-1 [Ly-6A/E]), conjugated with phycoerithrin (PE)(PharMingen), 2B8 (anti-c-kit), conjugated with FITC (PharmMingen), V-18(anti-IL-3R alfa) (Santa Cruz Biotechnology, Inc.), M-20 (anti-EpoR)(Santa Cruz Biothechnology, Inc. ), M-20 (anti-G-CSFR) (Santa CruzBiothechnology, Inc.), anti-rabbit IgG-fluorescein conjugated (FITC).Anti-nNOS mAb, anti-macNOS mAb, anti-eNOS mAb and polyclonal anti-nNOSantibodies were purchased from Transduction Laboratories. Polyclonalanti-nNOS antibodies were also purchased from Zymed.

[0131] BrdU Labeling

[0132] To identify cells in S phase mice were injected intraperitoneallywith 50 ug/ml 5-Bromo-deoxyuridine (BrdU) (Beckton-Dickinson) once a dayfor 5 days. Bone marrow cell smears were prepared and fixed with 4%formaldehyde. BrdU-labeled S phase nuclei were visualized afterdenaturing DNA in 2 M HCl, 0.5% Triton for 2 hours, and in; cubationwith fluorescein-conjugated antibodies to 5-BrdU (Becton dickinson assuggested by the manufacturer. Samples were analyzed on a Ziess Axiphotflourescent microscope. For nuclei visualization, smears were stainedwith DAPI, a flourescent DNA stain (Molecular Probes), at 1 mM.

[0133] Tunel

[0134] Analysis of apoptosis was performed on bone marrow cell smearsfixed 15 minutes with 4% formaldehyde in PBS by TUNEL assay (BoerhingerManheim) as suggested by the manufacturer.

[0135] NADPH Diaphorase

[0136] NADPH-diaphorase staining was performed essentially as described(Dawson, T. M., et al., Proc. Natl. Acad. Sci. USA, 88:7797 (1991) andHope, B. T., et al., Proc. Natl. Acad. Sci. USA, 88:2811 (1991)) withminor modifications. cells were fixed in 3.7% paraformaldehyde for 1hour, washed in PBS, and incubated for 60 min at 37° C. in the stainingsolution containing 1 nM NADPH, 0.025% Nitroblue tetrazolium salt and0.3% Triton.

[0137] Peripheral blood was analyzed using standard methods. Leukocyteswere counted in the hematocytometer and in methanol-fixed and Giemazastained smears of peripheral blood.

[0138] Results

[0139] Inhibition of NOS activity in experimental animals

[0140] In order to increast rhe number of stem and early progenitorcells in the bone marrow the following protocol was used:

[0141] a) a mixture of two NOS inhibitors, L-NAME (concentration 300 mgper kg of body weight) and ETU (concentration of 30 mg per kg of bodyweight) was introduced intraperitoneally twice a day.

[0142] b) the treatment was stopped after 3, 4, 5, 7 days or 9 days adnthe presence of specific markers was analyzed in the bone marrow by FACSanalysis 1, 2, 3, 5, 7, etc. days fter the cessation of treatment.

[0143] This protocol has dramatically increased the proportion of earlyprogenitor cells in the bone marrow. At first the increase is minimal oractually reversed compared to the control animals. However, several daysafter cessation of treatement, the proportion or progenitor cells (ckit-positive) becmae much igher than in control animale which receivedthe saline solution.

[0144] The content of c-kit-positive cells in the bone marrow wasincreased from 5.1% to 23/9%. The content of IL-receptor-positive cellswas increased from 4.3% to 25%. The content of Sca-positive cells in thebone marrow as increased from 1.7% to 5.1%. The content of Sca- andc-kit-positive cells (Sca⁺c-kit⁺cells) in the bone marrow was increasedfrom 0.4% to 1.48%.

[0145] The highest increased for c-kit in the bone marrow was at day 1after cessation of treatment with NOS inhibitors which was ongoing for 9days. The highest increase for IL3-R in the bone marrow was at days 2-3after cessation of treatment with NOS inhibitors which was going for 9days. The highest increase for Sea in the bone marrow was at days 1-2after cessation of treatment with NOS inhibitors, also ongoing for 9days.

[0146] Similar changes were observed in the spleens of the treatedanimals. The highest increase for c-kit in the spleen was at days 3-4after cessation of treatement with NOS inhibitors which was going for 9days. The highest increase for IL3-R in the spleen was at days 3-5 aftercessation of treatment with NOS inhibitors which was going for 9 days.The highest increase for Sca in the spleen was at days 1-3 aftercessation of treatment with NOS inhibitors which was going for 9 days.Thus, the changes in the content of the early progenitor markers in thespleen were following the kinetics of maturation of hematopoieticprecursor cells in the bone marrow.

[0147] Together, these results demonstrate that a novel protocol forinhibition of NOS is especially effective for enrichment of bone marrowand spleen in early hematopoietic progenitors.

[0148] NOS Inhibitors and Changes in the Peripheral Blood

[0149] The composition of the peripheral blood in the animals aftertreatment with NOS inhibitors was followed. It was found that thecontent of neutrophils dramatically increased and 5 days afterterminating NOS inhibitors injection it was increased 4.5 fold comparedwith the control animals, whereas 7 days after termination, neutrophilcontent was increased 7.4 fold compared with the control. Thisdemonstrayted that the progenitor cells whose content in the bone marrowwas increased by treatment with the mixture of two NOS inhibitors,successfully proceed through hematopoietic differentiation and areintroduced in the peripheral blood as mature granulocytes. Importantly,immature precursors in the peripheral blood were not observed, whichsuggests that treatment with NOS inhibitors does not induce neoplastictransformation of the bone marrow.

[0150] NOS Inhibition and DNA Synthesis

[0151] To evaluate the effect of NOS inhibition on DNA synthesis in bonmarrow incorporation of BrdU into the nuclei of bone marrow cells wastested. For this, mice were treated for 7 days with a mixture of two NOSinhibitors, L-NAME and ETU as described before, except that animals didnot receive gamma irradiation and bone marrow transplantation. For thelast 5 days of treatment animals received daily injection of BrdU at 50mg/kg intraperitoneally. Bone marrow was isolated and smears wereprepared, fixed using 4% formaldehyde and processed using anti-BrdUantibody as described in Materials and Methods. The proportion ofBrdU-labeled cells was significantly higher (up to 10 fold) compared tothe control bone marrow. This indicates that treatment with NOSinhibitors has a direct effect on DNA synthesis in the hematopoieticcells in bone marrow.

[0152] NOS Inhibition and Apoptosis

[0153] The test whether prolonged treatment with NOS inhibitors affectsthe rate of programmed cell death in bone marrow cells, the number ofapoptotic cells in the preparation of bone marrow cells was tested. TheTUNEL approach was used, thus revealing the cells with intenselyfragmented DNA, a hallmark of apoptosis, at the same time using DAPIstaining to visualize the nuclei of all cells in the preparation. Theresults indicated that prolonged treatment with the mixture of twoinhibitors, L-NAME and ETU, does not affect programmed cell death inbone marrow preparations. This indicates that manupulation of NOSactivity in the animals after BMT, although having profound effects ondifferentiation and maturation of hematopoietic cells, doed not affectthe extent of programmed cell death in BM cells, further supporting thefeasibility of application of NOS inhibitors for therapy.

[0154] Retransplantation of Bone Marrow and Spleen Hematopoietic Cellsto the Secondary Recipients

[0155] As described herein, treating animals with NOS inhibitors afterbone marrow transfer dramatically increased the number of cells whichexpress stem and progenitor cells' markers. This indicates that as aresult of treatment with NOS inhibitors, bone marrow becomes enriched instem and early progenitor cells. However, it was possible that thisprocedure only affected the levels of expression of the markers, or itincreased the proportion of immediate progenitor cells but not ofmultipotent hematopoietic stem cells. To directly demonstrate thatinhibition of NOS activity results in an increase in the number of stemcells, the proportion of colony forming units in the bone marrow andspleen of experimental animals was tested by transferring the bonemarrow or spleen cells to secondary recipients.

[0156] To obtain experimental animals, mice were irradiated at a dose8.2-9, 0 Gy and injected intravenously 3-5×10⁴ or intraperitoneally1×10⁶ with bone marrow cells from syngeneic donors. Immediately afterbone marrow transplanting experimental mice received intravenous orintrapertoneal injections of a mixture of two NOS-inhibitors: L-NAME andETU. NOS inhibitor injections were repeated twice daily for 9 days. Micein the control group were injected with saline solution. After 9 days,injections were suspended. Experimental and control mice were sacrificed1 or 3 days after termination of NOS inhibitor injections. To evaluatemultipotent stem cell (CFUs) and pre-CFUs content 3×10⁴ bone marrowcells or 1×10⁸ spleen cells from experimental or control mice weretransferred into secondary irradiated recipient mice intravenously. Inaddition, aliquots of bone marrow or spleen cells from the experimentalanimals were tested by FACS for the presence of c-kit or Sca-1 moleculesor both of them on the cell surface. After 8 and 12 days the secondaryrecipients were sacrificed and the hematopoietic colonies in theirspleen were counted. The number of day-12 spleen colonies was 3.5 foldgreater in mice which received bone marrow cells from experimentalanimals (primary recipients, treated with mixture of two NOS-inhibitorsfor 9 days and left untreated for 1 day) (3.5±0.22) than from thecontrol primary recipients which received saline solution (1.0±0.15). Incontrast, the number of day-8 spleen colonies was 2.9 fold less insecondary recipients which received bone marrow cells from experimentalmice (1.50±0.23), than in the secondary recipients which received bonemarrow cells from control mice (4.38±0.76). This indicates that underthese experimental conditions the number of more primitive CFUs-12 inbone marrow of primary recipients is increased, whereas the number ofmore committed CFUs-8 is decreased. The increase after 12 dayscorresponded to the increase in the number of c-kit-positive cells inbone marrow from the primary recipients as determined by FACS analysis.

[0157] When similar experiments were performed using only one of the NOSinhibitors, either ETU or L-NAME injected for 8-12 days, the number ofday-8 and day-12 colonies in the spleen of the secondary recipients wasincreased 1,7-2.5 fold, which indicates that treatment with a mixture oftwo NOS inhibitors is a) more effective than the use of either inhibitoralone, and b) leads to a specific enrichment of bone marrow populationwith more primitive stem cells (CFUs-12).

[0158] When experiments were performed with spleen cells transplantedfrom the primary to the secondary recipients, the number of day-12spleen colonies was 1.5 fold greater in the secondary recipients whichreceived spleen cells from the experimental animals (primary recipients,treated with mixture of two NOS inhibitors for 9 days and left untreatedfor 3 days) compared to the secondary recipients which received spleencells from mice of the control group injected with saline solution.

[0159] Together, these experiments directly demonstrate that inhibitionof NOS in the bone marrow of primary recipients led to an increase inthe proportion of multipotent hematopoietic stem cells (CFUs). Theseresults indicate that exposure to inhibitors of NOS thrapeutically usedto increase the proportion of stem cells in the bone marrow.

[0160] Furthermore, these experiments indicate that different inhibitorsof NOS activity and their combinations can be used for enrichment withhematopoietic stem and progenitor cells. Moreover, they suggest that, inaddition to bone marrow, a similar approach can be applied to stem andearly progenitor cells of blood such as umbilical cord vein blood orperipheral blood and other tissues of the organism to increase theircontent.

EXAMPLE 5 Xenopus Neurogenesis

[0161] To test the effect of exogenously supplying NO to the developingtadpole brain, pieces of the slow-release matrix Elvax impregnatedeither with S-nitroso-N-acetylpenicillamin (SNAP, a widely used donor orNO which releases NO upon hydrolysis), or with saline as a control wereinserted into the brain ventricle of state 45 tadpoles. At 1 or 3 daysafter implantation the following parameters in the optic tectum wereanalyzed: the total number of cells (as measured by the number of BrdUincorporation), the number of apoptotic cells ( as measured by TUNELassay), the realative numbers of BrdU- and TUNEL-positive cells per 10⁵total cells, the size of the optic tectum, and the cell density in thetectum (Table 5). The data show that exogenously supplied NO drasticallydecreases the number of proliferating cells at day 1 to only 5% of thecontrol value (Table 5). At 3 days the effects of NO on cellproliferation are less pronounced, likely reflecting the completehydrolysis of the NO donor and the cessation of NO release. Both thecell density and the total number of cells were markedly reduced at 1and 3 days. An increase in programmed cell death in response to NOlikely contributes to the decrease in the total number of cells in thebrain. However, the TUNEL data show that both the absolute and therelative numbers of apoptotic cells were decreased in the presence ofSNAP indicating that programmed cell death cannot be invoked to explainthe effects of SNAP on the change in the total cell number.

[0162] The close proximity of proliferating cells and NOS-expressingcells in the optic tectum and data from the NO donor experiments aboveare consistent with the proposed antiproliferative role of NO duringXenopus brain development. To test the causative role of NO in cellcycle arrest of neuronal precursors and to determine whether productionof NO is necessary for cell cycle arrest and subsequent differentiationof neuronal precursors in the developing Xenopus brain, NOS activity inthe brain was inhibited by using Elavax matrix impregnated with a NOSinhibitor, either L-NAME or ETU, or with saline as a control. Pieces ofimpregnated matrix were inserted into the brain ventricle of stage 45tadpoles. After 3 days of treatment the brains were examined for changesin the patterns of cell division and differentiation, cell number, andoverall size and morphology.

[0163] Both NOS inhibitors significantly increased the numbers of cellsin S phase compared to untreated or saline treated control animals ateach of the time-points tested (Table 6). Surplus BrdU-positive cellswere numerous in the proliferative zone of the optic tectum. They werealso present in more lateral and rostral regions of the tectum, whichare normally occupied by postmitotic differentiated neurons.

[0164] To assess whether this surplus of cells in the S phase in animalstreated with NOS inhibitors increased the total number of cells in thebrain, sections were stained with DAPI to reveal the cell nuclei. Incontrol brains, cells in the optic tectum occupy a crescent extendingalong the medial and caudal edges of the tectal lobe. Only a fewscattered cell bodies are located within the neuropil region where inputaxons mingle with tectal cell dendrites. Brains of animals treated withNOS inhibitors have significantly more DAPI-stained cells than untreatedor saline-treated control animals (Table 6), indicating that surpluscells in the S phase successfully completed the cell cycly by division.Extra cells distorted the lamination pattern in the optic tectum, formedectopic islands, and filled the tectal neuropil.

[0165] A decrease in programmed cell death likely contributes to theincrease in cell numbers following treatment with NOS inhibitors. Thus,the number of apoptotic cells was assayed with the TUNEL asssay to testthe effect of inhibitin of NOS on programmed cell death in thedeveloping tadpole brain. While the number of BrdU-positive cells andthe total number of DAPI-stained nuclei significantly increased afgter a3 day exposure to L-NAME, there was no corresponding change in theabsolute or even the relative number or distribution of TUNEL-positiveapoptotoc cells (Table 6). This demonstrates that excessiveproliferation of neuronal precursors due to inhibition of NOS was notaccompanied by an immediate and significant change in cell death,indicating that programmed cell death did not significantly contributeto the increase in the total cell number which was the most importantconsequence of NOS inhibition.

[0166] To test whether the increased number of cells affects the size ofthe tadpole brain, the changes in the volume of the dorsal midbrain andin the tectal cell density after inhibition of NOS were determined(Table 6). The cell density in the optic tectum after 3 days oftreatment showed no significant difference between treated and controlanimals, however after 7 days of treatment the cells were more denselypacked. A significant increase of the midbrain volume was seen indrug-treated animals as compared to saline treated controls using bothNOS inhibitors, ETU and L-NAME (29% increase over control at day 3 andan 85 increase at day 7 for L-NAME, and 23% increase at day 7 for ETU;see Tble 6).

[0167] To determine whether the cells generated in experiments using NOSinhibitors are able to undergo neuronal differntiation and expressneuronal markers, the distribution of tectal neurons expressing thepan-neuronal markers β-II tubulin and N-CAM was determined. In bothcontrol and L-NAME-treated brains, β-II tublin and N-CAMimmunoreactivity was concentrated in the tectal neuropil, however bothantibodies also label differentiated tectal cell bodies. Importantly,the staining pattern suggest that surplus cells in L-NAME treated brainsdifferentiate and express neuronal antigeens. Moreover, the antibodystaining revealed a gross distortion of the overall organization of theoptic tectum. The cell body region of the treated brains was thicker,where as the greatly disorganized neuropil was more loosely andehaotically packed with neurites, and the border between the neuropiland the cell body region was markedly irregular.

[0168] Antibodies to Islet-1 stain a subset of ventral motoneurons inchich whech are committed very early during vetebrate brain development.NOS inhibition did not affect the number and the distribution pattern ofIslet-1-expressing cells, although the increase in the size of the braindue to exposure to NOS inhibitor was apparent. This is consistent withthe early differentiation of these neutrons occuring before theinhibitors were applied. These data indicate that NOS inhibition caninfluence the development of cells in the brain within a specific windowof their development when they exit the cell cycle and begin todifferentiate. Together, the results indicate that NO is an essentialnegative regulator of neuronal precursor proliferation during vetebratebrain development.

[0169] Methods

[0170] Animals, NOS inhibitors, and NO donors. Albino Xenopus laevistadpoles were obtained by human chorionic gonadotrophin induced matingsand raised under standard condigions. At state 45⁵, animals wereanesthetized in 0.025 3-aminobernzoic acid (MS-222; Sigma) and a tinypiece (100 μm²×30 μm) of slow release Elvax plastic polymer (Dupont) wasinserted into the tectal ventricle through an incision made in theoverlying skin with a 30 gauge needle. Elvax was prepared with stockconcentrations of the NOS inhibitors, 2-ethyl-2thiopseudourea (ETU,Sigma) or L-nitro-arginine methy ester (L-NAME, Sigma) or the NO donorS-nitroso-acetylpenicillamnin (SNAP, Sigma) prepared as a 1:10 ratio ofchemical to polymer matrix. Low molecular weight molecules, includingL-NAME, are released at a constant rate over a period up to 30 days asthe lyophilized Elvax matrix slowly hydrates. Preliminary esxperimentswith a range of inhibitor stoch concentrations (10 mM, 100 mM and 1L-NAME and 1 nM, 10 mM and 100 mM ETU) indicated that 1 M L-NAME and 10nM ETU increased BrdU incorporation without any signs of toxicity ot theanimals. Similarly, SNAP was tested at a range of concentrations and wasfound to be effective at 300 mM. Control animals were treated with Elvaximpr3egnated with saline (0.1 M phosphate buffer).

[0171] Histochemistry and antibodies. Immunocytochemistry withwholemount preparations and sections of the tadpole was performed asdescribed. Monoclonal antibodies to β-II tubulin, N-CAM (developed by U.Rutishauser), and Islet-1 (developed by T. M. Jessell) were obtainedfrom the Developmental Studies Hybridoma Bank developed under theauspices of the NICHD and maintained by the University of Iowa,Department of Biological Sciences, Iowa City, Iowa 52243. Anti-mouseantibody conjugated to fluorescein (Bochringer Manheim) was used as asecondary antibody. Specimens were visualized and photographed underfluorescence or Normarski optics on a Zeiss Axiophot. For nucleivisualization, brain sections were stained with DAPI, a fluorescent DNAstain (Molecular Probes) at 1 μM. To identify cells in S phase tadpoleswere injected with 50 μg/ml 5-Bromo-deoxyuridine (BrdU)(Beckton-dickinson). After two hours survival, animals were fixed in3.7% paraformaldehyde for 2 hours and then in 70% ethanol overnight.BrdU-labled S phase nuclei were visualized after denaturating DNA in 2NHCl, 0.5% Triton for 2 hrs, and incubation with flourescein-conjugatedantibodies to BrdU (Becton Dickinson) as suggested by the manufacturer.Analysis of apoptosis diring tadpole brain development was performed on20 μm brain sections by TUNEL assay (Boehinger Manheim) as suggested bythe manufacturer. Samples were analyzed on a Zeiss Axiophot flourescentmicroscope and NOran confocal microscope. 3 sequential matching dorsal20 μm sections, representing most of the optic tectum, of each animalwerecollected and used to determine the sections' volume, cell number(after DAPI staining), number of BrdU positive cells and number ofTUNEL-positive cells. Each sample of the brain (3 sections) representedapproximately ⅕ of the whole midbrain. At least 5 animals were analyzedfore each experiment. TABLE 5 NO donor SNAP decreases volume, celldensity, and total cell number in the tadpole brain. control, 1d SNAP,1d change control, 3d SNAP, 3d change cell number 5020 +/− 82  3983 +/−606  −20.7% 9125 +/− 194   5012 +/− 594** −45.1% brain volume 78.4 +/−4.8  66.2 +/− 6.4  −15.6% 116.5 +/− 6.2  80.1 +/− 8.6* −31.3% mm³ × 10⁻⁴cell density 64.4 +/− 5.3  62.6 +/− 14.1 −2.8% 78.6 +/− 3.0   62.3 +/−1.7** −20.8% (cells per mm³ × 10⁻⁴) BrdU-positive cells 312 +/− 25  12+/− 2  −96.2% 479 +/− 32  316 +/− 22  −34.0% (total) BrdU-positive cells62  3 −95.2% 52 63 +21.1% per 10³ cells TUNEL-positive cells 192 +/− 15 138 +/− 12  −28.2% 167 +/− 14  82 +/− 10 −51%   (total) TUNEL-positivecells 38 34 −10.5% 18 16 −11.1% per 10³ cells # significant for the cellnumber (p = 0.003, F = 43.3), brain volume (p = 0.027, F = 11.7), andcell density (p = 0.009 and F = 21.9).

[0172] TABLE 6 NOS inhibitors increase volume, cell density, and totalcell number in the tadpole brain. control, 3d L-NAME, 3d change control,7d L-NAME, 7d change ETU, 7d change cell number 3644 +/− 158  5136 +/−339* +40.9% 8373 +/− 928  13742 +/− 510**   +64.1% 12870 +/− 690* +53.7% brain volume 52.7 +/− 3.8  67.8 +/− 9.1* +28.6% 99.4 +/− 4.9 107.4 +/− 3.7    +8.0% 122.3 +/− 4.3*  +23.0% mm³ × 10⁻⁴ cell density 70+/− 7  78 +/− 11 +11.4% 83 +/− 6  128 +/− 10* +54% 105 +/− 3*  +25.5%(cells per mm³ × 10⁻⁴) BrdU-positive cells 137 +/− 3  507 +/− 17 +270%   149 +/− 15  428 +/− 9  +187%  550 +/− 50  +269%   (total)BrdU-positive cells 37 98 +160%   17 31 +182% 42 +247%   per 10³ cellsTUNEL-positive 120 +/− 5  173 +/− 9  +44.1% 81 +/− 7  128 +/− 9  +58%184 +/− 7  +128%   cells (total) TUNEL-positive 32 33 +3%  10   9.0 +10%14 +40.0% cells per 10³ cells # after 3 days of treatment with L-NAME (p= 0.016, F = 15.9) and 7 days of treatment with L-NAME (p = 0.007, F =25.7) or ETU (p = 0.018, F = 15.1), differences in brain volume weresignificant after 7 days of treatment with ETU (p = 0.024, F = 12.4),and differences in cell density - after 7 days of treatment with L-NAME(p = 0.015, F = 16.9) or ETU (p = 0.027, F = 11.5).

EXAMPLE 6 Adult Rat Neurogenesis

[0173] Various isoforms of NOS are expressed in the developing nervoussystem of mammals (Bredt, D. and Snyder, S. H., Neuron, 13:301-313(1994)). Moreover, in the adult mammalian brain, NOS is present in cellswhich bear the characteristics of the neural stem cells (e.g., can formneurospheres, a hallmark of multipotential neural stem cell) (Wang etal., Cell Tissue Res. 296, 489-497 (1999)). Thus, the distribution ofNOS in the developing and adult brain is consistent with theantiproliferative role of NO during mammalian neurogenesis.

[0174] To Test the role of NO as a negative regulatro of cellproliferation in the mammalian brain, NOS activity in the rat brain wasinhibited. The solution of NOS inhibitors was introduced in the lateralventricles of the adult rat brain using osmotic micropumps. 50 mMsolution of L-NAME, D-NAME, or saline as a control was injected over thetime course of 7 days. 4 days after treatment rats were injected with 60μg/g solution of 8-bromo-deoxyurdine (BrdU) 7 times at 12 hourintervals. Animals were sacrificed and brain sections were analyzed forthe number of BrdU-positive cells using anti-BrdU antibodies(Beckton-Dickinson). Treatment with NOS inhibitors increased the numberof BrdU-positive cells in the subventricular zone, rostral migratorystream, and hippocampus by 52.8%, 39.3%, and 12%, respectively. Anincrease in the number of BrdU-positive cells was also observed in thecortex, corpus callosum and other areas of the brain. Together, thesedata indicate that NO acts as a negative regulator of cell proliferationin the adult rat brain and that it is possible to increase the extent ofneurogenesis in the adult brain by blocking the production of NO.

EXAMPLE 7 Mouse Hematopoiesis

[0175] NOS expression in the bone marrow. Various isoforms of Nosynthase are produced in the most of the types of tissues in humans androdents. To examine the presence of NOS protein or mRNA isoforms in themouse bone marrow, the preparations of the isolated bone marrow wereanalyzed by Western blots and RT-PCR. NOS protein isoforms were notdetected when probing the total bon marrow lysates with antibodies toneuronal, endothelial, or inducible forms of NOS, probably due to theirlow content in the preparations of the total bone marrow. However, mRNAof each NOS form were detected when using RT PCR. The RT PCR signalswere reliable and specific for each of the NOS genes tested, since theywere reproducibly obtained with several combinations of primers for eachgene, produced by DNA fragments of the expected nucleotide sequence, andgenerated DNA fragments of the expected size after digestion withrestriction endonucleases. These results indicate tha all three genescoding for NOS isoforms are expressed in the bone marrow.

[0176] In vitro cultivation of murine bone marrow cells. To test therole of NO signaling in hematopoietic maturation of bone marrow cells invitro, 1×10⁷ of bone marrow cells from C57B1/6 mice were added to flaskswith preliminary formed adhesive stromal feeder laryer. The feeder layerwas prepared from the bone barrow of syngeneic mice plated 3 weeksbefore the experiment (1 femur per 50 ml flask). NOS inhibitors wereadded and the appearance of the hematopoiesis markers was tested atweekly intervals. After 2 weeks of treatment with NOS inhibitors thenumber of c kit positive cells was 2.7 fold higher in cultures treatedwith a mixture of 0.5 mM FET and 5 mM L-NAME and 1.6 fold higher incultures treated with 1 nM L-NAME as compared with the control cultures.Together, this indicates that inhibition of NOS activity can be used toincrease the number of hematopoietic stem and early progenitor cells invitro.

[0177] In vitro cultivation of human umbilical cord blood cells. To testthe role of NO signaling in hematopoietic maturation of humanhematopoietic cells, human umbilical cord blood (HUCB) cels were used.When HUCB cells were grown in the presence of NOS inhibitors, thecontent of CD34 positive cells was increased 1.8 fold after growing for1 to 2 weeks in the presence of 1 mM L-NAME and 2.6 fold in the presenceof 0.5 mM ETU and 5 mM L-NAME. When HUCB cells were permitted to growfor 3 weeks before adding NOS inhibitors, the increase in the content ofCD34-positive cells was even higher, reaching 4.7 fold for 1 nM L-NAMEafter 2 weeks of treatment.

[0178] When similar experiments were performed with adhesive stromalfeeder layer prepared from the bone marrow of NOD/SCID mice, theincrease in the content of CD34-positive cells was 1.7 and 1.4 foldafter 1 and 2 weeks, respectively.

[0179] Together, these experiments indicate that inhibition of NOSactivity can be used to increase the number of human hematopoietic stemand early progenitor cells in vitro.

[0180] Transplantation of human umbilical cord blood cells in toNOD/SCID mice. To test the potential of NOS inhibitors to affect humanhematopoietic cells in vivo, 10-25×10⁶ mononuclear cells from HUCB weretransplanted into sublethally irradiated NOD/SCID mice. Animals weretreated with NOS inhibitors or saline as a control. The number ofCD34-positive cells was increased 1.8, 5.5, and 7.4 fold after treatmentwith 30 mg/kg ETU and 300 mg/kg L-NAME for 6, 14, and 20 days,respectively, as compared with the control. These experiments suggestthat NO acts to control proliferation and maturation of humanhematopoietic cells in vivo.

[0181] Equivalents

[0182] While this invention has been particularly shown and describedwith reference to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spririt and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

What is claimed is:
 1. A method of increasing in a mammal a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, comprising contacting the hematopoietic tissue with multiple doses of at least one inhibitor of nitric oxide synthase, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
 2. The method of claim 1 wherein the inhibitor is contacted with the hematopoietic tissue for a period of days selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.
 3. A method according to claim 1 wherein the step of contacting is carried out ex vivo.
 4. A method according to claim 3 further comprising transplanting the hematopoietic tissue having an increased population of hematopoietic stem cells into a mammal in need thereof.
 5. A method according to claim 1 wherein the differentiation of erythroid cells is prevented.
 6. A method according to claim 1 wherein the differentiation of myeloid cells is prevented.
 7. A method according to claim 1 further comprising contacting the hematopoietic tissue with at least one hematopoietic growth factor selected to induce differentiation of a selected hematopoietic stem cell population.
 8. A method according to claim 1 wherein the inhibitor of nitric oxide synthase is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2-thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine.
 9. The method for treating a mammal to increase a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue of the mammal, comprising contacting the hematopoietic tissue of the mammal with multiple doses of at least one inhibitor of nitric oxide synthase, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
 10. The method of claim 9 wherein the inhibitor is contacted with the hematopoietic tissue for a period of days selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.
 11. A method according to claim 9 further comprising contacting the hematopoietic tissue with at least one hematopoietic growth factor selected to induce differentiation of a selected hematopoietic stem cell population.
 12. A method for treating a mammal to increase a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue of the mammal, comprising the steps of: a) obtaining hematopoietic tissue which is to be transplanted into the mammal; b) contacting the hematopoietic tissue to be transplanted with multiple doses of at least one inhibitor of nitric oxide synthase; c) transplanting the hematopoietic tissue of step (b) into the mammal to be treated, thereby providing the mammal with hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
 13. The method of claim 12 wherein the inhibitor is contacted with the hematopoietic tissue for a period of days selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.
 14. A method according to claim 12 further comprising: d) treating the mammal with an enhancer of nitric oxide synthase after transplanting the hematopoietic tissue.
 15. A method according to claim 12 further comprising: d) treating the mammal with an inhibitor of nitric oxide synthase after transplanting the hematopoietic tissue.
 16. A method of increasing a population of progenitor blood cells which are capable of undergoing normal hematopoiesis, differentiation and maturation, comprising contacting progenitor cells of blood with multiple doses of at least one inhibitor of nitric oxide synthase, thereby increasing the population of progenitor blood cells.
 17. The method of claim 16 wherein the inhibitor is contacted with the blood for a period of days selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.
 18. A method according to claim 16 wherein the progenitor cells of the blood are obtained from hematopoietic tissue selected from the group consisting of: bone marrow, umbilical cord vein blood, peripheral blood, fetal liver and long term hematopietic cell cultures.
 19. A method according to claim 16 wherein the inhibitor of nitric oxide synthased is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine.
 20. A method of increasing a population of dividing cells in a tissue of a mammal comprising contacting the cells with multiple doses of at least one inhibitor of nitric oxide.
 21. A method according to claim 20 wherein the inhibitor is an inhibitor of nitric oxide synthase.
 22. A method according to claim 20 which results in an increase in the size of an organ with which the tissue is associated.
 23. A method of decreasing a population of cells in S phase in a tissue of a mammal and inducing differentiation of the cells, comprising contacting the tissue with multiple doses of at least one enhancer or nitric oxide.
 24. A method according to claim 23 wherein the enhancer is an enhancer of nitric oxide synthase.
 25. A method to claim 23 which results in a decrease in the size of an organ with which the tissue is associated.
 26. A method of regenerating tissue in an adult mammal comprising contacting a selected tissue with multiple doses of at least one inhibitor of nitric oxide, thereby inhibiting differentiation and inducing proliferation of cells of the tissue, then contacting the selected tissue with a compound which inhibits proliferation and induces differentiation.
 27. The method of claim 26 wherein the compound which inhibits proliferation and induces differentiation is selected from the group consisting of: nitric oxide, a growth factor, or a combination of both.
 28. The method of claim 26 wherein the inhibitor of nitric oxide is an inhibitor of nitric oxide synthase.
 29. The method of claim 26 which results in an increase in the size of an organ with which the tissue is associated.
 30. A method according to claim 26, wherein the tissue is selected from the group consisting of blood, skin, bone, digestive epithelium, fat tissue, bone marrow stroma, cartilage and tendon.
 31. A method of repopulating an organ or tissue having normally nondividing cells comprising contacting a selected organ or tissue with multiple doses of at least one inhibitor of nitric oxide, thereby inhibiting differentiation and inducing proliferation of cells of the organ or tissue, then contacting the selected organ or tissue with a compound which inhibits proliferation and induces differentiation.
 32. The method of claim 31 wherein the compound which inhibits proliferation and induces differentiation is selected from the group consisting of: nitric oxide, a growth factor, or a combination of both.
 33. The method of claim 31 wherein the inhibitor of nitric oxide is an inhibitor of nitric oxide synthase.
 34. The method of claim 31 which results in an increase in the size of the organ.
 35. A method according to claim 31 wherein the organ or tissue is selected from the group consisting of muscle and nerve fibers.
 36. A method of producing a subpopulation of hematopoietic cells in hematopoietic tissue comprising the steps of: a) contacting the hematopoietic tissue with multiple doses of at least one inhibitor of nitric oxide synthase, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation; and b) contacting the hematopoietic tissue with at least one hematopoietic growth factor selected to induce specific differentiation of the hematopoietic stem cell population, thereby producing a subpopulation of hematopoietic tissue.
 37. The method of claim 36 wherein the inhibitor is contacted with the bone marrow for a period of days selected for the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.
 38. A method according to claim 36 wherein the inhibitor of nitric oxide synthase is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2-thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine.
 39. A method of increasing a population of cells in S phase in a tissue of a mammal, comprising contacting the tissue with multiple doses of at least one inhibitor of nitirc oxide.
 40. A method according to claim 39 wherein the cells in S phase can be used in gene therapy. 